Compositions and methods for tissue engineering, tissue regeneration and wound healing

ABSTRACT

In accordance with certain embodiments of the present disclosure, a kit is described. The kit includes primed living cells joined to and at least partially within a three-dimensional hydrogel structure and an isolated polypeptide having the carboxy-terminal amino acid sequence of an alpha Connexin, or a conservative variant thereof, wherein the polypeptide does not include the full length alpha Connexin protein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 2P20RR016434-06awarded by the National Institutes of Health. The government retainscertain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/896,196 having a filing date of Oct. 1, 2010 (allowed and issued asU.S. Pat. No. 8,409,603), which is based on and claims priority to U.S.Provisional Application Ser. No. 61/247,806, filed Oct. 1, 2009 and U.S.Provisional Application Ser. No. 61/316,550, filed Mar. 23, 2010, whichare incorporated by reference herein in their entirety.

BACKGROUND

Body organs display complex shapes, surfaces and internal structures,including blood vessels. Coupled to morphological complexity, thecomponent tissues of organs possess dynamic contractile and mechanicallyresponsive elements such as sphincter muscles. The ability to accuratelyrecapitulate naturally occurring complexities of shape, internalstructure and functionality is a key goal of tissue engineering. Currentapproaches to this problem include the use of scaffolds to generate invivo like environments for cell growth or “organ printing” wherepoint-by-point extrusion from a modified ink-jet printer has enabledcells to be precisely arranged into three-dimensional tissue-likestructures.

Technologies based on culture scaffolds or organ printing rely onapplication of straightforward engineering principles. To date, thefield has been less successful in harnessing self-organizing processesoccurring during embryonic development to elaborate useful biologicalstructure in vitro.

Similarly, a tissue-engineered approach for wound healing attempts topromote regenerative and scar-free healing. In this regard, stem cellsare increasingly being utilized as part of regenerative therapies. Todate, conventional regenerative therapies based on stem cells involvethe introduction of dispersed cells into a diseased organ with littleregard for the status of these randomly introduced cells. Currenttherapies often show marginal, variable, or even controversialpropensity to promote regenerative and scar free healing. As such,improvements to available compositions and methods would be beneficial.

The epithelial-mesenchymal transition (EMT) precedes virtually everycellular differentiation in the embryo. For example, during humandevelopment one of the first rounds of EMT occurs as part ofgastrulation, in which the 3 embryonic germ layers differentiate.Subsequent rounds of EMT within germ layers occur as a prelude tomorphogenetic processes as diverse as neurogenesis, myogenesis, andvasculogenesis.

The precise role of EMT is unclear. EMT resembles a priming step thatprecedes morphogenetic events. Progenitor (i.e., stem) cells form stablecell-cell junctional contacts, synchronizing cytoskeletal organization,polarity, and to some extent proliferative activity. The cells thendetach, transforming into invasive mesenchymal cells in a coordinatedprogression toward terminal differentiation.

The present disclosure describes a unique and non-obvious series ofsteps to stimulate an EMT-like state in cells in a culture dish(referred to as EMT-priming or activation) to provide: a) microtissuecompositions (e.g., toroids of EMT-primed/activated cells) with medicaluses in tissue engineering and b) regenerative healing methods andcompositions as described in the subsequent text of this disclosure.

SUMMARY

In accordance with certain embodiments of the present disclosure, a kitis described. The kit includes primed living cells joined to and atleast partially within a three-dimensional hydrogel structure and anisolated polypeptide having the carboxy-terminal amino acid sequence ofan alpha Connexin, or a conservative variant thereof, wherein thepolypeptide does not include the full length alpha Connexin protein.

In another embodiment of the present disclosure, a method for forming ahydrogel structure in which living cells are joined thereto isdisclosed. The method includes placing living cells on athree-dimensional hydrogel structure. The method further includespermitting epithelialization of the cells in which the cells attach tothe surface of the hydrogel structure, undergo epithelial mesenchymaltransition, and penetrate the surface of the hydrogel structure.

In still another embodiment of the present disclosure, a method ofpromoting healing following tissue injury in a subject is disclosed. Themethod includes administering to the subject's injury primed livingcells which are joined to and at least partially within athree-dimensional hydrogel structure and an isolated polypeptidecomprising the carboxy-terminal amino acid sequence of an alphaConnexin, or a conservative variant thereof, wherein the polypeptidedoes not comprise the full length alpha Connexin protein.

Other features and aspects of the present disclosure are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention is set forth inthe specification with reference to the following figures.

FIG. 1 illustrates examples of toroids generated from EMT-primed lensepithelial cells; a-c) a control series of 3 toroids generated inregular media, d-f) a control series of 3 toroids generated in mediacontaining a control peptide and g-h) a series of 3 toroids generated inmedia containing a an ACT1 peptide in combination with Tgfb1.

FIG. 2 illustrates examples of gel in which lens epithelial cells (i.e.,non-EMT primed) have been premixed into collagen (right hand panels). Notoroid forms in this case (upper right hand panel) and the cells withinthe gel are disorganized (lower right hand panel). A toroid is generatedfrom EMT-primed lens epithelial cells (upper left hand panel);immunoconfocal optical sectioning reveals the cellular composition ofthe toroidal ring within the gel (lower left hand panel).

FIG. 3 illustrates a high power view of phalloidin-stained aligned cellsin a toroid.

FIG. 4 illustrates a) examples of “apertured” spheroids formed byadjustment of the initial geometry of constraint of the gel over a 48hour time course, b) if the cells are pre-mixed into the gel,morphogenesis into a spheroid does not proceed.

FIG. 5 illustrates hypothetical examples of how toroids can be used toa) enhance wound closure of a slow-healing diabetic wound, b) beassembled iteratively into the branched tubular scaffold of anartificial blood vessel.

FIG. 6 illustrates toroid use in wound healing.

FIG. 7 illustrates the layout of the equally sized treatment/controlwounds on either side of the dorsal midline of a rat.

FIG. 8 illustrates that combinatorial treatment including a bone-marrowderived stem cells (BMSC) toroid promotes dramatic reduction in skinscarring.

FIG. 9 illustrates a histological demonstration that the combinatorialtreatment including the BMSC toroid (but not when BMSCs are mixed in thegel) dramatically reduces scar tissue formation and promotesepidermal/dermal regeneration.

FIG. 10 illustrates a quantitative analysis that shows that a treatmentof a BMSC toroid and ACT1 (but not when BMSCs are mixed in the gel withACT1) provides statistically significant reduction in scar tissueformation following skin wounding.

FIG. 11 illustrates a quantitative analysis showing that a combinatorialtreatment of a BMSC toroid and TGFb3 (but not when BMSCs are mixed inthe gel with TGFb3) provides significant reduction in scar tissueformation following skin wounding.

FIG. 12 illustrates a histological demonstration that the combinatorialtreatment including the BMSC toroid with TGFb3 (but not when BMSCs aremixed in the gel with TGFb3) dramatically reduces scar tissue formationand promotes epidermal/dermal regeneration.

FIG. 13 illustrates the heart injury model in rat developed to testregenerative repair of heart by the disclosed invention.

FIG. 14 illustrates the ocular corneal burn injury model developed totest regenerative repair of injured cornea by the disclosed invention.

DETAILED DESCRIPTION

Reference now will be made in detail to various embodiments of thedisclosure, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the disclosure, notlimitation of the disclosure. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present disclosure without departing from the scope or spirit ofthe disclosure. For instance, features illustrated or described as partof one embodiment, can be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present disclosurecovers such modifications and variations as come within the scope of theappended claims and their equivalents.

The present disclosure describes a simple method for promptingself-organization of cells in gels into repeatable and geometricallydistinct microtissue units, including rings and hollow spheroids. Forinstance, the present disclosure describes methods for generatingtissue-engineered rings (also referred to herein as “toroids”) of cellsin vitro. The methods described herein provide a novel approach todirecting self-organizing morphogenetic principles that are intrinsic tothree-dimensional networks of cells. These have the potential to beiteratively combined to tissue engineer more complex structures. As aconsequence, the microstructure and function of toroids and otherstructures generated as described in the present disclosure closelyresemble naturally occurring complexities of tissues in livingorganisms, including humans. The methods described herein can also beadjusted to generate other useful objects, including spheroids.Spheroids are generated by using a square gel, rather than one ofcircular geometry. In other embodiments the starting gel geometry cantake a variety of forms and sizes including circular, square,rectangular trapezoid, or any regular of irregular two-dimensionalshape. The starting gel can also be in the form of a tube or any otherso constrained three-dimensional structure.

In another embodiment, the initial gel used to construct the tissueengineered composition can be a tubular tissue scaffold wherein the wallis comprised of polymer fibrils that are aligned in a helical patternaround the longitudinal axis of the tube where the pitch of the helicalpattern changes with the radial position in the tube wall.

In another embodiment, a pattern of stress or strain can be impartedstatically, dynamically or in combination on a gel containing cellsundergoing EMT-priming to achieve a desired cellular geometry.

In yet another embodiment self-organized toroids generated in accordancewith the present disclosure can be used in a tissue-engineered assistcomposition for wound healing, tissue engineering or regenerativehealing or as a scaffold for elaboration of a tissue, such as anartificial venous sphincter muscle. The present disclosure alsodescribes how toroids and other morphogenetically engineered structuresgenerated by the methods described herein can be iteratively stacked,fused or otherwise combined to generate engineered tissues of complexgeometry including synthetic blood vessels.

For example, tissue-engineered structures generated by the methodsdescribed herein such as toroids or other geometric structures can becombined with each other to form complex three-dimensional arrays, andif required, organ or organ like structures or other biologicalstructures such as a bone and the like. In general, thesethree-dimensional arrays include two or more layers separately appliedto a substrate, with subsequent layers applied to the top surface oflower layers. These tissue engineered organs and biological structureswould then be used as a therapy in the regenerative repair of, forexample, damaged, diseased or congenitally malformed organs and tissues.

In certain embodiments, the layers can combine or fuse followingplacement on the substrate or, alternately, remain substantiallyseparate and divided following application. Three-dimensional structurescan be formed in a variety of ways in accordance with the presentdisclosure. For example, three-dimensional arrays can be formed byplacing multiple layers onto the substrate using an organ printingdevice or using a novel device that iteratively combines toroids tobuild up complex three-dimensional structures by layering onetissue-engineered structure on another in a manner contemplated as beingsimilar to a ham slicer working in reverse. The tissue engineeredstructures in the layers can be retained in the gel upon iteration intoa three dimensional array and or largely separated from the gel, such aswould occur if the composition had been generated in thermo-gellinghydrogel.

It is sometimes desired when three-dimensional structures are generatedthat that any subsequent cell growth is substantially limited topredefined sectors of the structure. Thus, to inhibit or promote growth,promote differentiation, or promote or inhibit apoptosis inside oroutside of a predefined region, compounds, devices or the like can beapplied to specific regions of substrate to inhibit cell growth,differentiation, or cell death, thus forming a boundary within thestructure. Some examples of suitable compounds for this purpose include,but are not limited to, growth factors, cytokines, drugs, agarose,poly(isopropylN-polyacrylamide) gels, and the like. Such factors can bepresent in some layers and not in others to generate the desiredthree-dimensional structure.

In one example of such embodiments, the process of generating a boundarycan be employed to form a multi-layered, three-dimensional tube such asa blood vessel (e.g, FIG. 5 b). In such embodiments would be desired togenerate a growth restricted toroidal layer of endothelial cells withina larger diameter toroid of smooth muscle and then stack such compositesto generate a tube. Growth restriction within the endothelial layerprior and during fusion of the layers of the structure would act toprevent the tube that eventually formed from becoming occluded.

In addition to layer-by-layer assembly, three-dimensional arrays canalso be formed by placing a single layer of the tissue-engineeredcompositions onto a substrate. In such embodiments, this layer would beallowed to grow in culture and develop into a single or multi-layeredstructure. In one simple example, a toroid of endothelial cells can beplaced on a substrate within a larger diameter toroid of smooth musclecells. These composite rings can then be stacked, fused or otherwisecombined to generate a blood vessel like tube. In another example, theapproach would be used to generate extended two-dimensional layers offused unitary compositions such as the case if artificial skin was beingprepared. In yet a further example, extended two dimensional sheetswould be combined with unitary tissue-engineered composition or othersimilarly manufactured sheet to build up large scale three dimensionalstructure such as would be required to iterate a large organ such as aliver or distinct biologic structures such as an ear cartilage or abone. These tissue-engineered structures would then be used as a therapyin the regenerative repair of, for example damaged, diseased orcongenitally malformed organs or tissues.

Very generally, one aspect of the present disclosure (i.e., in thegeneration of an individual tissue engineered composition) is amulti-step process in vitro.

The process includes initiation, which requires placement of dispersedsuspensions of living cells on a flexible three-dimensional culturehydrogel that are constrained by adherence to a rigid surface (e.g., theplastic bottom and sides of a circular tissue culture well, or thelike). The hydrogel can be composed of collagen in one example (e.g.,FIG. 1). The hydrogel can also be composed of keratins, fibrins,chitosans, glycosaminoglycans, elastins, matrigel, carrageenan,chondroitin sulfate, gelatin, pectin, alginate or gels based oncombinations of these naturally occurring components), other naturallyoccurring or synthetic components comprising the gel can includefibronectins, laminins, proteoglycans, hyaluronan, glc-nac,matricellular proteins (e.g., periostins and related polypeptides, CNN1,thrombospondins), collagen or elastin mimetics or combinations thereof,or synthetic hydrogel components (e.g. polyurethane hydrogel, PEGhydrogel, polyacrylate hydrogel, polyvinyl alcohol, sodium polyacrylate,acrylate polymers and copolymers, carboxymethyl or carboxyethylcellulose or combinations thereof), hybrid hydrogels synthetic ornaturally occurring components (e.g., gels composed of collagen andpolyethylene glycol (PEG)) and naturally occurring, synthetic or hybridhyrogels functionalized with drugs, sugars, peptides, particles or otherattached or integrated factors and the like.

Other examples of hydrogel materials that can be used alone or incombination with other gel composite materials include polylacticglycolic acid-ethylene glycol-lactic glycolic acid, poly hydroxybutyrate, polypropylene fumarate-co-ethylene glycol, polylacticacid-ethylene glycol-lactic acid, polyethylene glycol-lacticacid-ethyleneglycol, polymethylmethacrylate-co-hyroxyethylmethacrylate,polyethylene glycol-bis-lactic acid-acrylate, polyethyleneglycol-butylene oxide-terephtalate, polylactic glycolic acid-c-serine,polyhydroxypropylacrylamide-g-peptide, polyethyleneglycol-g-acrylamide-co-vemine, polyethylene glycol/-cyclodextrins,polyvinylacetate/vinylalcohol, polyhydroxyethylmethacrylate/Matrigel,polyN-vinyl pyrrolidone, polyacrylonitrile-co-allil sulfonate,polyethylene glycol dimethacrylate-sulfate,polyethyleneglycol-co-peptides), polybiscarboxy-phenoxy-phospazene,alginate-gpolyethylene oxide-propylene oxide-ethylene oxide,polyacrylamide, polyN-isopropyl acrylamide-co-acetic acid,polyN-isopropyl acrylamide-co-ethylmethacrylate, chitosan-g-polyethyleneoxide-propylene oxide-ethylene oxide, hyaluronic acid-g-N-isopropylpolyacrylamide, alginate-acrylate and collagen-acrylate.

In another example, a hydrogel that can be activated to become fluid bylowering its temperature, to about 25° C. or below (i.e., roomtemperature). For example, such gels would be used to generate thetissue-engineered composition and then the gel depolymerized bytemperature lowering so that the cellular elements of the compositionthat had been prompted by the methods described herein, to self-organizeinto the desired geometry, can be isolated from the hydrogel and thenfused or otherwise combined to form new compositions as described hereinto generate complex three- and two dimensional structures. One exampleof such multi-dimensional compositions would be tissue engineered organor organ-like structures used in the regenerative repair of, for exampledamaged, diseased or congenitally malformed organs.

An example of a thermo-gel is a polymer of a meth-acrylamide derivativeand a hydrophilic monomer. A further example of a thermogel hydrogelpolymer is a biodegradable polymer containing a polyethylene glycol(PEG) block linked to biodegradable polyester. Other examples are gelsbased on pluronic acid (PLA) that become liquid at 4 C but formhydrogels at 37 C. These thermogels can also be mixed with othernaturally occurring on synthetic hydrogel materials or contain othercompounds that improve their performance in generating and maintainingthe tissue engineered composition. For example, addition of steroidalhydrocortisone to PLA thermogel improves the viability of cellscontained within the gel (Khattak et al. Tissue Eng. 2005 May-June;11(5-6):974-83, incorporated by reference herein).

The next step of the multi-step sequence required to generate thecomposition is what is termed epithelialization. Epithelializationrefers to a period of culture in which cells to attach to the surface(i.e., the substrate) of the cultured hydrogel and initiate cell-matrixand cell-cell interactions. The epithelial mesenchymal transition (EMT)is one of the most fundamental morphogenetic mechanisms in extantmulticellular organisms. During EMT, epithelial cells adherent to asubstrate surface transform to assume a migratory and invasivemesenchymal phenotype. In developing vertebrates, cells undergoing EMTinclude those contributing to gastrulation, neurogenesis, skeletalmyogenesis, cardiomyogenesis, and vasculogenesis. EMT is a necessaryprelude to basic differentiation processes occurring in the human embryoand also has been shown to have assignments in cancer and in the genesisof stem cells. Thus in certain aspects, the steps of the presentdisclosure embody reverse engineering of a developmental process toprovide a novel composition in a culture dish, a composition, device andmethod with therapeutic assignments.

A further period of culture takes place in which cells on the culturegel surface undergo an epithelial-mesenchymal-like transformation,invading the gel interior and remodeling cell-cell interactions as thisinvasive migration proceeds. Three-dimensional cellularization takesplace as still a further period of culture occurs in which the cellsproliferate, migrate, and differentiate stable new patterns ofthree-dimensional cell-cell and cell matrix interaction within thecultured gel. Activation of cells to form the unitary microtissuecomposition is further contributed to by a precisely timed release ofthe cellularized three-dimensional gel from attachment to itsconstraint. Lastly, morphogenesis occurs as a further period of culturewherein the cells in the gel undergo a process of re-organization into aring of aligned and electromechanically coupled contractile cells orother self-organized structure such as a spheroid dependent on the novelinventive steps disclosed herein for directing this morphogenesis-likeprocess in vitro.

Certain aspects of the individual steps and sequence of steps describedherein are both necessary and non-obvious aspects of the presentdisclosure. For instance, if cells are mixed directly into the gelthereby omitting certain other steps, morphogenesis of the cells into aprovided complex tissue structure does not proceed and the usefulness ofsuch compositions in methods of regenerative healing as described laterin this disclosure are reduced. In one example when stem cells are“pre-mixed” into a collagen gel, as opposed to being activated in vitroas described herein, this “mixed” stem cell composition is not observedto provide benefits to wound healing, scar reduction and tissueregeneration provided by the tissue-engineered composition of EMT-primedstem cells in the disclosure provided herein. Furthermore, EMT-primedBMSCs and other stem cell types can improve immune tolerance of otherengrafted tissues, organs, or bioengineered structures. For instance,the literature describes BMSCs having this effect (Ghannam S, Bouffi C,Djouad F, Jorgensen C, Noël D. Immunosuppression by mesenchymal stemcells: mechanisms and clinical applications. Stem Cell Res Ther. 2010Mar. 15; 1(1):2., incorporated by reference herein).

It should be understood, however, that various modifications to theprocess described herein are contemplated in accordance with the presentdisclosure. For example, the present disclosure contemplates thattissue-engineered toroids of smaller and larger diameter (e.g., ˜1 mm to3-5 cm and in a larger example structures of about up to 10 cm) will begenerated by modifying certain parameters including the diameter of theculture well, cell number at initiation, and the physical and chemicalproperties of the gel. Similarly, different constructs including oblongrings, irregular rings and even hollow shapes such as spheres, ovoidsand other irregular geometries will be generated by modifications thatinclude adjustment of the initial pattern of rigid constraint,adjustment of parameters described previously, and/or manipulation ofthe timing of gel release and adjustment of the physical and chemicalregularity of the gel. As an example, FIG. 4 a shows hollow spheroids,each with a small aperture that were generated in a square-sided well.

Similarly, while a gel-based method is simple and convenient, thepresent disclosure contemplates that synchronization and activation ofepithelial-mesenchymal-like transformation is achievable in receptivecells by other means including via genetic, pharmaceutical, and/orchemical manipulations. It is understood that the present disclosureprimarily demonstrates the need for recapitulation of synchronizationand activation in EMT-like morphogenesis in vitro, prior to uses thatinclude generation of complex three-dimensional structures frommicrotissue units or transplantation into a wound.

Various cell types are contemplated by the present disclosure includingepithelial cells, mesenchymal cells, endothelial cells, bone marrowcells, spleen cells, lymphatic cells, stem cells, progenitor cells(natural or induced), all embryonic, postnatal and adult derivatives ofendoderm, mesoderm or ectoderm cells, vascular cells, muscular cells,mesenchymal cells, hematopoietic cells, fibroblasts, myofibroblasts,osteogenic cells, fibrogenic cells, neurogenic cells, myogenic cells,smooth muscle cells, cardiomyocytes, and combinations thereof.

Other examples include Keratinizing epithelial cells: e.g., Epidermalkeratinocyte, Epidermal basal, Nail bed basal, and Hair matrix cells;Wet stratified barrier epithelial cells: e.g., epithelial cells ofstratified squamous epithelium of cornea, tongue, oral cavity,esophagus, anal canal, distal urethra and vagina and basals cells of thesame tissues; Gland cells; e.g., Exocrine secretory epithelial cells;e.g., Hormone secreting cells, including Anterior pituitary,Intermediate pituitary cell, secreting melanocyte-stimulating hormone,Magnocellular neurosecretory, Gut and respiratory tract, Thyroid glandcells, Parathyroid gland cells, Adrenal gland, Leydig, Juxtaglomerular,Macula densa, Peripolar cell and Mesangial cells; Metabolism and storagecells: e.g., Hepatocyte, White fat and Brown fat cells and lipocytes;Barrier function cells of Lung, Gut, Exocrine Glands and UrogenitalTract, Epithelial cells lining closed internal body cavities: e.g.,Blood vessel and lymphatic vascular endothelial fenestrated cell, Bloodvessel and lymphatic vascular endothelial continuous cell, Blood vesseland lymphatic vascular endothelial splenic cell, Choroid plexus cell,Pigmented ciliary epithelium cell of eye, Nonpigmented ciliaryepithelium cell of eye, Corneal endothelial cell; Ciliated cells withpropulsive function: e.g., Respiratory tract ciliated cell;Extracellular matrix secretion cells: e.g., Ameloblast epithelial,Planum semilunatum epithelial cell, Loose connective tissue, Corneal,Tendon, Bone marrow and Other nonepithelial fibroblasts, Pericytes,chondrocytes, Osteoblast/osteocyte, Osteoprogenitor cells, Hyalocyte ofvitreous body, and Stellate cells; Contractile cells: Skeletal muscleand Satellite cells (stem cell) and Heart muscle stem cells, Smoothmuscle cells, Myoepithelial cells, Blood and immune system cells; e.g.,leukocytes, monocytes, cell of the nervous system e.g., Astrocytes,Neurons, and Oligodendrocytes, Anterior lens epithelial cell, Pigmentcells, Germ cells, Nurse cells and Interstitial cells. Progenitor andembryonic cells as well as committed progenitors of forementioned celltypes provided in this paragraph.

These and other cell types will be able to sufficiently model theprocess described herein such that the aforementioned structures willalso form from these cell types. It is furthermore anticipated that twoor more different cell types can be mixed in the same gel todifferentiate the aforementioned structures comprised of one or morecell types.

It is also anticipated that any cell type receptive to synchronizationand activation by the provided method should be a sufficient substratefor inducing the regenerative healing benefit described herein. Stemcells are probably especially receptive, as such bone-marrow derivedstem cells (BMSCs) can be substituted by other stem cell types includingtotipotent, omnipotent, pluripotent, multipotent, oligopotent andunipotent stem cell types, including embryonic, fetal, and adults stemcells, amniotic stem cells and other stem cells derived from the variousstem cell niches and fluids found within or emanating from the humanbody, mesenchymal stem cells, tissue and lineage specific stem cells andinduced progenitor stem cells. Other differentiated cell types shouldalso provide benefit following induction in vitro by the present method,particularly if it is combined with a regimen that reverts these cellsto induced pluripotent stem cells (iPS) or iPS-like state.

In addition, targeted treatments with drugs, cytokines, chemicals, andother bioactive substances will provide a further way of modulating anddirecting the differentiation of the aforementioned structures (e.g.FIGS. 1 g-h). For instance, a treatment of skin wounds with a toroid ofBMSCs and ACT1 significantly enhances regenerative healing and inhibitsscarring over that occurring for treatments with a BMSC toroid alone orthe peptide alone. In another example, treatment of skin wounds with atoroid of BMSCs and TGF-beta3 significantly enhances regenerativehealing and inhibits scarring over that occurring for treatments with aBMSC toroid alone or the peptide alone. TGF-B3 and/or ACT1 can be usedcoincident with or after the injury or can be introduced to a site onthe subject prior to a surgery or any anticipated tissue-disruptingprocedure (e.g., non-surgical dermabrasion) in order to precondition thesite for the purpose of scar reduction, improved tissue structure andfunction and regenerative repair. The pre-conditioning can be at orrepeated at 1, 2, 3, 4, 5, 6 hours or any other interval up to 24 hours.The pre-conditioning can be at or repeated at 1, 2, 3, 4, 5, 6 or anyother interval up to 48, 72, and 96 hours and up to a week. Followingpreconditioning and surgery further post-treatment with the invention orconstituents (TGF-B3 or ACT1) can be undertaken. BMSCs not induced toorganize into the provided compositions did not show propensity topromote regenerative and scar-free healing. The present composition andmethod, involving the artificial generation of a primitiveembryonal-like tissue in vitro and the transplantation of this tissueinto a wound, embodies a novel therapeutic approach that convenientlyrecapitulates embryonal scarless healing in the postnate or adult.

The present disclosure contemplates combination of the providedcomposition with treatments or pre-conditioning treatments other thanACT1 and TGF-beta3 known to improve healing and/or reduce scarringincluding osteopontin, platelet-derived growth factor (PDGF),transforming growth factor and beta, TGFb or Cx43 antisense or peptidescan be of significant benefit. Other molecules that are contemplated foruse with the present disclosure include bone morphogenetic proteins(BMP), epidermal growth factors (EGF), erythropoietins (EPO), fibroblastgrowth factors (FGF), platelet derived growth factors (PDGFs), ligandsfor the seven transmembrane helix family, granulocyte-colony stimulatingfactor (GCSF), granulocyte-macrophage colony-stimulating factor (GMCSF),growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF),hepatoma derived growth factor (HDGF), human growth hormones (HGH),interleukins (IL), insulin growth factors (IGF), insulin growth factorbinding proteins (IGFBP), myostatins (GDF-8), nerve growth factors (NGF)and other neurotrophins, thrombopoietins (TPO), vascular endothelialgrowth factors (VEGF), transglutmaninases, caveolins, matricellularproteins (e.g., periostin, CCNs, thrombospondins), osteopontin,canonical (e.g., Wntl, Wnt3a) and non-canonical WNTs (e.g., Wnt5a,Wnt11), planar cell polarity pathway signaling components, interleukins,tumor necrosis factors (TNFs), Notch-Delta, hyaluronin and relatedmolecules, Hyaluronic synthetic enzymes (e.g., HAS2, HAS3), relaxins,acetylcholine, chitosan, DMSO, N-acetyl-glucosamine, catecholamines,poly unsaturated fats, estrogens and related/derivative molecules,androgens and related molecules, inhibitors of collagen processing(e.g., prolyl 4-hydroylase, C-proteinase and lysyl hydroxylase, HRTpeptidases), ZP123, AAP10, rotigaptide, RXP-E and related compoundsbinding the Cx43 CT, NADPH oxidases, factors effecting connective tissuegrowth factors (CTGFs), endothelins, and angiotensins, complementproteins, Protein Kinases (e.g., PKC-alpha, PKC-beta, PKC-epsilon,PKC-zeta and other PKC), bioactive fragments or polymers of thesemolecules (e.g, CT sequences from Protein Kinases), genetic or cellularvectors producing these molecules, binding proteins, molecules targetingthe receptors or downstream signal transduction mediators andcombinations thereof. As these molecules and their different familymembers can have opposing effects in different circumstances ligands,agonists (activating factors) and antagonists (or inhibiting factors) ofthese molecules will be used in the present disclosure.

The polypeptide used with the disclosed tissue engineered invention canbe any polypeptide comprising the carboxy-terminal most (CT-most) aminoacids of a Connexin, wherein the polypeptide does not comprise thefull-length Connexin protein. ACT1 peptide used in one embodiment of thecomposition is one example of such a CT-most peptide from a connexin.

Connexins are the sub-unit protein of the gap junction channel, which isresponsible for intercellular communication (Goodenough and Paul. Nat.Rev Mol Cell Biol. 2003 April; 4(4):285-94, incorporated by referenceherein). Connexin channels as well as structurally related molecules canalso act as single membrane channels or hemichannels. Connexin moleculescan also act as regulatory molecules of signal transduction pathways. Inone example, it has been shown that Cx43 can modulate the TGF-betasignaling pathway via a targeting of SMADs, downstream regulators ofTGF-beta signaling (Dai et al., Mol Biol Cell. 2007 June; 18(6):2264-73,incorporated by reference herein).

The carboxy-terminus (CT)-most sequence of connexins is a key regulatorydomain. For example, the CT-most amino acid sequences of Connexins arecharacterized by distinct and conserved features. This preservation ofstructure is consistent with the ability to form characteristics 3Dshapes, interact with Cx43 in intra and intermolecular associations,interact with multiple other proteins, interact with lipids andbiomembranes, interact with nucleic acids including RNA, transit and/orblock membrane channels and provide consensus sequences for proteolyticcleaving, cross-linking, ADP-ribosylation, glycosylation andphosphorylation. Thus, ACT1 interacts with a domain of a protein thatnormally mediates the binding of said protein to the CT-most sequence ofa Connexin. For example, the scaffolding protein ZO-1 interacts with theCT-most domain of Cx43 (Toyofuku et al., J Biol. Chem. 1998 May 22;273(21):12725-31, incorporated by reference herein). It is consideredthat this and other proteins interact with the CT-most sequence ofConnexins and further interact with other proteins forming a complex ofmultiple proteins. The polypeptide used with the tissue engineeredcomposition can inhibit the operation of a molecular machine, such as,for example, one involved in regulating the aggregation of Cx43 gapjunction channels from hemichannels.

In a further example, nephroblastoma overexpressed protein (NOV)interacts with a Cx43 CT-most domain (Fu et al., J Biol. Chem. 2004279(35):36943-50, incorporated by reference herein). NOV is amatricellular protein found in the matrix external to cells that has keyfunctions in wound healing. Connexin based peptides thus haveassignments that occur outside of the cell or at the external membranesurface of the cell.

In one example defining CT-most connexin peptide there is a conservedproline or glycine residue in Connexins consistently positioned some 17to 30 amino acids from the carboxyl terminal-most amino acid. Forexample, for human Cx43 a proline residue at amino acid 363 ispositioned 19 amino acids back from the carboxyl terminal mostisoleucine. In another example, for chick Cx43 a proline residue atamino acid 362 is positioned 18 amino acids back from the carboxylterminal-most isoleucine. In another example, for human Cx45 a glycineresidue at amino acid 377 is positioned 19 amino acids back from thecarboxyl terminal most isoleucine. In another example for rat Cx33, aproline residue at amino acid 258 is positioned 28 amino acids back fromthe carboxyl terminal most methionine. The polypeptide used with thedisclosed invention can thus comprise the CT-most 4 to 30 amino acids ofthe Connexin, including the c-terminal most 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,amino acids of the Connexin.

The CT most amino acids of a Connexin can be flanked by non-CT-mostamino acids. Examples of the flanking amino acids are provided herein.An example of non-CT-most Connexin amino acids is the carboxy-terminal20 to 120 amino acids of human Cx43. Another example would be thecarboxy-terminal 20 to 120 amino acids of human Cx45. Another examplewould be the carboxy-terminal 20 to 120 amino acids of chick Cx45.Another example would be the carboxy-terminal 20 to 120 amino of humanCx37. Another example would be the carboxy-terminal 20 to 120 aminoacids of rat Cx33.

CT-most peptides retain function when flanked with non-Connexinpolypeptides of up to at least 239 amino acids. Indeed, as long as thesequence is maintained as the free carboxy terminus of a givenpolypeptide, and the peptide is able to access its targets and be ableto be used with the present disclosure. Thus, polypeptides exceeding 239amino acids in addition to the CT-most peptide can function in reducinginflammation, promoting healing, reducing scarring and promoting tissueregeneration in association with the disclosed invention followinginjury.

The sequence of the polypeptide used with the present disclosure can befrom any Connexin. Thus, the Connexin component of the polypeptide canbe from a human, murine, bovine, monotrene, marsupial, primate, rodent,cetacean, mammalian, avian, reptilian, amphibian, piscine, chordate,protochordate or other Connexin or conservative variant thereof.

Thus, the provided polypeptide can comprise a component of a Connexinselected from the group including Connexin 47, Connexin 46.6, Connexin30.2, Connexin 30.2, Connexin 31.9, Connexin 26, Connexin 32, Connexin44, Rat Connexin 33, Connexin 36, Connexin 37, Connexin 39, Connexin40.1, Connexin 38, Connexin 39.9, Connexin 40, Connexin 43, ZebrafishConnexin 43, Connexin 43.4, Connexin 44.2, Connexin 44.1, Connexin 45,Connexin 46, Connexin 56, Connexin 39.9, Connexin 49, Connexin 50,Connexin 59, Connexin 32, Connexin 26, or other Connexin. These examplesand other amino acid sequences for connexins, structurally relatedmolecules known as pannexins and conservative variants thereof are knownin the art.

The 20-30 CT-most amino acid sequence of most Connexins arecharacterized by a distinctive and conserved organization. Thisorganization can include a class/type II PDZ binding motif (Φ-x-Φ;wherein x=any amino acid and Φ=a Hydrophobic amino acid (aa) and next tothis motif, Proline (P) and/or Glycine (G) aas; a high frequencyphospho-Serine (S) and/or phospho-Threonine (T) residues; and a highfrequency of positively charged Arginine (R), Lysine (K) and negativelycharged Aspartic acid (D) or Glutamic acid (E) aas. For many Connexins,the P and G residues occur in clusters next to the CT PDZ binding motif.The S and T phospho-amino acids of many Connexins also are often inclustered, repeating sequences. This organization is the case for Cx43,where 90% of 20 CT-most aas are the latter seven amino acids.

The CT-most peptide of Cx43 is highly conserved from humans to fish(e.g., compare Cx43 sequences for humans and zebrafish). In a furtherexample, CT-most sequence of Cx45 is highly conserved from humans toavians. In yet another example, the sequence of Cx36 is highly conservedfrom primates to fish

Thus, in one aspect, the polypeptide used in association with thepresent disclosure comprises one, two, three or all of the amino acidmotifs selected from the group consisting of 1) a type II PDZ bindingmotif, 2) Proline (P) and/or Glycine (G) hinge residues; 3) clusters ofphospho-Serine (S) and/or phospho-Threonine (T) residues; and 4) a highfrequency of positively charged Arginine (R) and Lysine (K) andnegatively charged Aspartic acid (D) and/or Glutamic acid (E) aminoacids). In another aspect, the provided polypeptide comprises a type IIPDZ binding motif at the carboxy-terminus, Proline (P) and/or Glycine(G) hinge residues proximal to the PDZ binding motif, and positivelycharged residues (K, R, D, E) proximal to the hinge residues.

PDZ domains were originally identified as conserved sequence elementswithin the postsynaptic density protein PSD95/SAP90, the Drosophilatumor suppressor dlg-A, and the tight junction protein ZO-1. Althoughoriginally referred to as GLGF or DHR motifs, they are now known by anacronym representing these first three PDZ-containing proteins(PSD95/DLG/ZO-1). These 80-90 amino acid sequences have now beenidentified in well over 75 proteins and are characteristically expressedin multiple copies within a single protein. Thus, in one aspect, theprovided polypeptide can inhibit the binding of a Connexin to a proteincomprising a PDZ domain. The PDZ domain is a specific type ofprotein-interaction module that has a structurally well-definedinteraction ‘pocket’ that can be filled by a PDZ-binding motif, referredto herein as a “PDZ motif”. PDZ motifs are consensus sequences that arenormally, but not always, located at the extreme intracellular carboxylterminus Four types of PDZ motifs have been classified: type I(S/T-x-Φ), type II (Φ-x-Φ), type III (Ψ-x-Ψ) and type IV (D-x-V), wherex is any amino acid, Φ is a hydrophobic residue (V, I, L, A, G,W, C, M,F) and Ψ is a basic, hydrophilic residue (H, R, K). (Songyang, Z., etal. 1997. Science 275, 73-77, incorporated by reference herein). Thus,in one aspect, the polypeptide used with the provided disclosure wouldcomprise a type II PDZ binding motif.

The Cx37 ACT-like sequence is GQKPPSRPSSSASKKQ*YV. Thus the carboxyterminal 4 amino acids of Cx37 conform only in part to a type II PDZbinding domain. Instead of a classical type II PDZ binding domain, Cx37has a neutral Q* at position 2 where a hydrophobic amino acid would beexpected. As such Cx37 comprises what can be termed a type II PDZbinding domain—like sequence. Nonetheless, Cx37 strictly maintains allother aspects of peptide organization including clustered serineresidues, frequent R and K residues and a P-rich sequence proximal tothe PDZ binding domain-like sequence. Given this overall level ofconservation of ACT-like organization in common with the other >70Connexins described herein, it is understood that the Cx37 CT functionsin the provided capacity in the disclosure described herein.

The Connexin Cx26 has no carboxyl terminal type II PDZ binding motif;about 30% of the carboxyl terminal most amino acids comprise S, T, R, Dor E residues; it has no evidence of motifs proximal to a type II PDZbinding motif or PDZ binding like motif containing clusters of P and Ghinge residues; and no evidence of clustered, repeat-like motifs ofserine and threonine phospho-amino acids. Cx26 does have three Lysine(K) residues, clustered one after the other near the carboxy terminus ofthe sequence.

As herein provided with the present disclosure, the unique functionalcharacteristics of this relatively short stretch of amino acidsencompass unexpected roles in reducing inflammation, promoting healing,reducing scarring and promoting regeneration of complex tissue structureand function following injury in tissues as diverse as skin and brain.Thus, in one aspect, the provided polypeptide comprises a type II PDZbinding motif (Φ-x-Φ; wherein x=any amino acid and Φ=a Hydrophobic aminoacid). In another aspect, greater than 40%, 50%, 60%, 70%, 80%, 90% ofthe amino acids of the provided polypeptide is comprised one or more ofProline (P), Glycine (G), phospho-Serine (S), phospho-Threonine (T),Arginine (R), Lysine (K), Aspartic acid (D), or Glutamic acid (E) aminoacid residues.

The amino acids Proline (P), Glycine (G), Arginine (R), Lysine (K),Aspartic acid (D), and Glutamic acid (E) are necessary determinants ofprotein structure and function. Proline and Glycine residues provide fortight turns in the 3D structure of proteins, enabling the generation offolded conformations of the polypeptide required for function. Chargedamino acid sequences are often located at the surface of folded proteinsand are necessary for chemical interactions mediated by the polypeptideincluding protein-protein interactions, protein-lipid interactions,enzyme-substrate interactions and protein-nucleic acid interactions.Thus, in another aspect Proline (P) and Glycine (G) Lysine (K), Asparticacid (D), and Glutamic acid (E) rich regions proximal to the type II PDZbinding motif provide for properties necessary to the actions ofpeptides in association with the present disclosure. In another aspect,the polypeptide comprises Proline (P) and Glycine (G) Lysine (K),Aspartic acid (D), and/or Glutamic acid (E) rich regions proximal to thetype II PDZ binding motif.

Peptides, peptide mimetics or conservative variants can be made tomodulate gap junction, hemichannel or other independent biologicalfunctions in association with the present disclosure that are based onthe amino-terminal, extracellular, cytoplasm loop and transmembranedomains of connexin family members can also be used to develop peptidegap junction, hemichannel of connexin based signal transductionmodulating agents. Such peptides can comprise from ˜3 to ˜30, forexample a 14 amino acid long sequence, or from ˜6 to ˜15 aminocontiguous amino acids of sequence, for example a 6 amino acid longsequence. In a further example, Cx43 mimetic peptidergic inhibitors ofCx43-based gap junction communication that can be used with the presentdisclosure include:

FEVAFLLIQWI, LLIQWYIGFSL, SLSAVYTCKRDPCPHQ E2,

VDCFLSRPTEKT, SRPTEKTIFII, LGTAVESAWGDEQ, QSAFRCNTQQPG,

QQPGCENVCYDK E1, VCYDKSFPISHVR E1.

Related sequences incorporating the extracellular loop domains thatmodulate gap junction, hemichannel or other independent biologicalfunctions can be found for all other connexin family members connexinsincluding Cx45, Cx40, Cx32, Cx26, Cx31 and all other known connexins andpannexins by those skilled in the art.

Genetic vectors (e.g., transfected cDNA, viral vectors and the like)expressing these sequences listed above or combinations of other factorswith said vectors and peptides are also contemplated to be used inassociation with the provided disclosure.

Phosphorylation is the most common post-translational modification ofproteins and is crucial for modulating or modifying protein structureand function. In one example Cx43 is phosphorylated at the serineresidue 368 (s368). Phosphorylation at s368 is associated withpreconditioning of the myocardium. Phosphorylation at s368 causeschanges in permselectivity of the Cx43 gap junction channel andhemichannels. Phosphorylation at s368 can promote communicationcompartments in tissues that inhibits spread of cell and tissue damageinto otherwise normal tissues adjacent to an injury. ACT1 can promotephosphorylation at s368. Aspects of protein structure and functionmodified by phosphorylation include protein conformation,protein-protein interactions, protein-lipid interactions, enzymaticfunction, protein-nucleic acid interactions, channel gating, proteintrafficking and protein turnover. Thus, in one aspect the phospho-Serine(S) and/or phospho-Threonine (T) rich sequences are necessary formodifying the function of the molecules, increasing or decreasingefficacy of the polypeptides in their actions. In another aspect, theprovided polypeptide comprise Serine (S) and/or phospho-Threonine (T)rich sequences or motifs. Exemplary phosphorylating agents are wellknown in the art and can include, TPA, Src or G protein-coupled receptorantagonists and agonists. Phosphorylation and dephosphorylation toinhibit, enhance or otherwise modify the activity of molecules used forthe purpose of the present disclosure are thus contemplated.

When specific proteins are referred to herein, variants, derivatives,and fragments are contemplated to be used with the present disclosure.Protein variants and derivatives are well understood to those of skillin the art and can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. These variants ordinarilyare prepared by site-specific mutagenesis of nucleotides in the DNAencoding the protein, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture. Techniquesfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known and include, for example, M13 primermutagenesis and PCR mutagenesis. Amino acid substitutions are typicallyof single residues, but can occur at a number of different locations atonce; insertions usually will be on the order of about from 1 to 10amino acid residues. Deletions or insertions preferably are made inadjacent pairs, i.e., a deletion of 2 residues or insertion of 2residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNA structureunless such a change in secondary structure of the mRNA is desired.Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Suchsubstitutions generally are referred to as conservative substitutions.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinations.Conservatively substituted variations of each explicitly disclosedsequence are included within the polypeptides provided herein.

Typically, conservative substitutions have little to no impact on thebiological activity of a resulting polypeptide. In a particular example,a conservative substitution is an amino acid substitution in a peptidethat does not substantially affect the biological function of thepeptide. A peptide can include one or more amino acid substitutions, forexample 2-10 conservative substitutions, 2-5 conservative substitutions,4-9 conservative substitutions, such as 2, 5 or 10 conservativesubstitutions.

A polypeptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a polypeptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods. An alanine scan can be used toidentify which amino acid residues in a protein can tolerate an aminoacid substitution. In one example, the biological activity of theprotein is not decreased by more than 25%, for example not more than20%, for example not more than 10%, when an alanine, or otherconservative amino acid (such as those listed below), is substituted forone or more native amino acids.

Further information about conservative substitutions can be found in,among other locations, in Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), all of which are incorporated by reference herein, and instandard textbooks of genetics and molecular biology.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also can be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, substition of seryl or threonyl residues with glutamyl andasparyl residues, methylation of the o-amino groups of lysine, arginine,and histidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco pp 79-86 [1983],incorporated by reference herein), acetylation of the N-terminal amineand, in some instances, amidation of the carboxyl-terminal.

It is understood that there are numerous amino acid and peptide analogs,which can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have differentsubstituents. The opposite stereoisomers of naturally occurring peptidesare disclosed, as well as the stereoisomers of peptide analogs. Theseamino acids can readily be incorporated into polypeptide chains bycharging tRNA molecules with the amino acid of choice and engineeringgenetic constructs that utilize, for example, amber codons, to insertthe analog amino acid into a peptide chain in a site specific way(Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller,Current Opinion in Biotechnology, 3:348-354 (1992); Ibba, Biotechnology& Genetic Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994), all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble polypeptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO— (These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CHH₂—S); Hann J. Chem. Soc Perkin Trans. 1307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. It isunderstood that peptide analogs can have more than one atom between thebond atoms, such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such (Such as what).Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) can be used to generate more stable peptides. Cysteineresidues can be used to cyclize or attach two or more peptides together.This can be beneficial to constrain peptides into particularconformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992),incorporated herein by reference).

Thus, for example, in the case of CT-most polypeptides, these sequencescan comprise a conservative variant. An example of a single conservativesubstitution within the sequence RPRPDDLEI is given in the sequenceRPRPDDLEV. An example of three conservative substitutions within thesequence RPRPDDLEV is given in the sequence RPRPDDVPV. Thus, theprovided polypeptide can comprise aa sequences.

It is understood that one way to define any variants, modifications, orderivatives of the disclosed genes and proteins herein is throughdefining the variants, modification, and derivatives in terms ofsequence identity (also referred to herein as homology) to specificknown sequences. Specifically disclosed are variants of the nucleicacids and polypeptides herein disclosed which have at least 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent sequenceidentity to the stated or known sequence. Those of skill in the artreadily understand how to determine the sequence identity of twoproteins or nucleic acids. For example, the sequence identity can becalculated after aligning the two sequences so that the sequenceidentity is at its highest level.

Another way of calculating sequence identity can be performed bypublished algorithms. Optimal alignment of sequences for comparison canbe conducted by the local sequence identity algorithm of Smith andWaterman Adv. Appl. Math. 2: 482 (1981), by the sequence identityalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443(1970), by the search for similarity method of Pearson and Lipman, Proc.Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection. These references are incorporatedherein by reference in their entirety for the methods of calculatingsequence identity.

The same types of sequence identity can be obtained for nucleic acidsby, for example, the algorithms disclosed in Zuker, M. Science244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710,1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989, which are hereinincorporated by, reference for at least material related to nucleic acidalignment.

Other non-peptidergic modulating agents that can be used in associationwith the present disclosure include, Fatty acids; oleic acid,arachidonic acid, and lipoxygenase metabolites; aliphatic alcohols;heptanol, octanol anesthetics; halothane, propofol, ethflurane, andthiopental; anandamide; arylaminobenzoate (FFA: flufenamic acid andlipophilic derivatives); 2′,5′-dihydroxychalcone;Chlorohydroxyfuranones;3-chloro-4-chloromethyl-5-hydroxy-2(5H)-furanone; dexamethasone;doxorubicin (and derivatives); eicosanoid thromboxane A(2) (TXA(2))mimetics; nitric oxide; Fenamates; Genistein; glycyrrhetinic acid(GA):18a-glycyrrhetinic acid and 18-beta-glycyrrhetinic acid, andderivatives thereof; lysophosphatidic acid; lindane; mefloquine;menadione; 2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaicacid; oleamide; PH, gating by intracellular acidification; e.g.acidifying agents; polyunsaturated fatty acids; quinidine; quinine; alltrans-retinoic acid; vitamin A and retinoic acid derivatives andtamoxifen.

Modulation of gap junctional intercellular coupling or betweenextracellular and the intercellular space by connexin/pannexinhemichannels, and effects of connexin domains on signal transduction andenzymatic pathways, chaperoning and transport of molecules, and effectson ECM molecular and cellular organization are within the scope ofcertain compounds and embodiments that will be used in the presentdisclosure.

It is contemplated that the aforementioned structures, including thoseof different size, shape regularity and cellular composition, can becombined to generate organ-like structures of complex shape, size andinternal structure, as illustrated in FIG. 4 b. It is also understoodthat the manufacture contemplated can involve an iterative mechanismthat is capable of combining stacks of the forementioned structures tobuild up increasing complexity of shape in sequential and repeatablemanner. The structures generated can be used as scaffolds upon which tobuild further complexity via addition of one or more cell types, matrixmolecules and other additives that enable the accretion of furtherbiological complexity upon the initial contractile scaffold.

The present disclosure can also assist in the healing of normal wounds,including those resulting from accidents, surgery or failure of healingof a surgical wound (e.g., a dehiscent wound).

For example, the certain aspects of the present disclosure will modulatecell migration and proliferation, thereby reducing inflammation,accelerating wound healing, reduce scarring and ultimately promoterepair, regeneration and restoration of structure and function in alltissues. The reduction in inflammation will speed-up wound closure andconsequently the process of wound healing. Healing of wounds,post-peptide application will involve significantly reduced fibrosis,consequently reduced scarring in skin wounds and fibrous patches ininternal tissue injuries, promoting tissue regeneration and restoringtissue and organ structure and function.

Further, the invented tissue engineered composition can be used to treatexternal wounds caused by, but not limited to scrapes, cuts, laceratedwounds, bite wounds, bullet wounds, stab wounds, burn wounds, sun burns,chemical burns, surgical wounds, bed sores, radiation injuries, allkinds of acute and chronic wounds, wounds or lesions created by cosmeticskin procedures and also ameliorate the effects of skin aging. Theactions of the present disclosure will accelerate wound healing in allkinds of external wounds and improve the cosmetic appearance of woundedareas, and skin subject to aging and disease. The disclosed inventionmay be provided directly, as a pre-treatment, as a pre-conditioning,coincident with injury, pre-injury or post-injury. The composition beused to treat internal injury caused by, but not limited to, disease,surgery, gunshots, stabbing, accidents, infarcts, ischemic injuries, toorgans and tissues including but not limited to heart, bone, brain,spinal cord, retina, peripheral nerves and other tissues and organscommonly subject to acute and chronic injury, disease, congenital anddevelopmental malformation and aging processes. Injury to internalorgans causes a fibrotic response, which leads to loss of structure andfunction in organ systems. In central nervous system (CNS) this responseto injury is mediated by astrocytes (fibroblast-like cells in the CNS)and thus will subsequently be referred to as an astrocytic response.Embodiments of the present disclosure will alleviate thisfibrotic/astrocytic response hence helping in repair and regeneration ofinjured tissues and restoration of tissue and organ structure andfunction.

Regenerative processes aided by the tissue engineered compositioninclude, but are not limited to internal and external injury,regeneration of tissues, organs, or other body parts, healing andrestoration of function following vascular occlusion and ischemia, brainstroke, myocardial infarction, spinal cord damage, brain damage,peripheral nerve damage, ocular damage (e.g., to corneal tissue), bonedamage and other insults to tissues causing destruction, damage orotherwise resulting from, but not limited to, injury, surgery, cancer,congenital and developmental malformation, and diseases causingprogressive loss of tissue structure and function, including but notlimited to diabetes, bacterial, viral and prion-associated diseases,Alzheimer's disease, Parkinson's disease, AIDs and other geneticallydetermined, environmentally determined or idiopathic disease processescausing loss of tissue/organ/body part structure and function. Inaddition, the compositions described herein can be administered withdrugs or other compounds promoting tissue and cellular regenerationincluding, but not limited to, trophic factors in processes including,but not limited to, brain, retina, spinal cord and peripheral nervoussystem regeneration (e.g., NGFs, FGFs, Neurtrophins, Neuregulins,Endothelins, GDNFs, BDNF. BMPs, TGFs, Wnts), as well as pre-conditioningfactors or stimuli e.g., hypoxia, norepinephrine, bradykinin,anesthetics, nitrate, ethanol, Alda-1, ALDH2 antagonists, PKC-epsilonagonists, exogenous ligands that activate opioid receptors (DPDPE,deltorphin II, methadone, SNC-80, BW373U86, DPI-287, DPI-3290) deliveredin a prospective pre-treatment prior to a surgery of other proceduredisrupting tissue in a subject.

A further embodiment of the present disclosure comprises the use oftissue engineered compositions to alleviate the symptoms of MultipleSclerosis (MS). MS is a chronic disease of the central nervous system.Pathologically, MS is characterized by the presence of areas ofdemyelination and T-cell predominant perivascular inflammation in thebrain white matter. The anti-inflammatory and regenerative properties ofthe treatment will help in the treatment of MS and other conditionssimilar to it.

The compositions of the present disclosure will help with conditionslike, but not limited to psoriasis, scleroderma, acne, eczema and otherdiseases of skin and connective tissues. Psoriasis, a chronic,inflammatory skin disease characterized by an uncontrolled shedding ofthe skin and afflicts millions of people throughout the world. Theeffects of the treatment on fibroblasts and inflammatory response of thetreatments, as stated in the claims above, will help alleviatePsoriasis. Eczema is characterized by painful swelling, oozing of theskin, bleeding cracks, severe scaling, itching and burning. As statedabove, the effects of the treatment on fibroblasts and inflammatoryresponse, combined with accelerated healing properties will relievesymptoms of eczema.

Said tissue engineered compositions will help with repair after cosmeticand/or clinical procedures involving, but not limited to, controlleddamage—e.g., corneal laser surgery, laser and dermabrasion/dermaplaning,skin resurfacing, and punch excision. Application of the treatmentimmediately after surgery or any cosmetic procedure will reduce oreliminate scarring. Uses of said composition will reduce keloid scarformation. Keloid scars are common in dark skin people of Asian,African, or Middle Eastern descent. Keloid scar is a thick, hypertrophicpuckered, itchy cluster of scar tissue that grows beyond the edges of awound or incision. Keloid scars are sometimes very nodular in nature,and they are often darker in color than surrounding skin. They occurwhen the body continues to produce tough, fibrous protein (known ascollagen) after a wound has healed. Application of the treatment willameliorate formation of Keloid or hypertrophic scars.

Additional uses of the tissue engineered compositions of the presentdisclosure will help correct other diseases and other conditions (e.g.,congenital and developmental defects, aging) associated withinflammatory response, fibrosis and connective tissue disorders.Fibrosis is a common condition noted after trauma to any bodily organ ortissue. Excessive fibrosis results in loss of structure and function andscarring at the trauma site. The treatment will reduce fibrosis andpromote regeneration, and restoration of structure and function.

Said compositions will modulate cell proliferation and can be used aloneor in association with drugs used in the treatment of uncontrolledproliferation (e.g., anti-cancer drugs) and procedures (e.g., radiationtherapy). Diseases of uncontrolled cell proliferation, or hyperplasias,are common health problems. Examples of diseases of cellover-proliferation include but are not limited to psoriasis, seborrhea,scleroderma, eczema, benign prostate hyperplasia, congenital adrenalhyperplasia, endometrial hyperplasia, squamous cell (vulvular)hyperplasia, sebaceous hyperplasia, Crohn's Disease, leukemia,carcinoma, sarcoma, glioma, and lymphoma. The compositions describedherein limit undesirable cellular proliferation and will thus improveprognosis of conditions associated with excessive cell proliferation.

The compositions of the present disclosure will have effects on cellmigration, proliferation and differentiation and thus will assist inpreventing metastasis. The compositions can be administered alone or inassociation with drugs or procedures used in the treatment of metastasislike but not limited to, Altretamine, Asparaginase, Bleomycin, Busulfan,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbesterol, Ethinylestradiol, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide,Goserelin, Hydroxyurea, Idarubicin, Ifosfamide, Leuprolide, Levamisole,Lomustine, Mechlorethamine, Medroxyprogesterone, Megestrol, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, pentastatin, Pipobroman, Plicamycin, Prednisone,Procarbazine, Streptozocin, Tamoxifen, Teniposide, Vinblastine,Vincristine. Metastasis is the spread of cancer from its primary site toother places in the body. Cell migration is the movement of cells fromone part of the body to another. The treatments of the presentdisclosure can effect cell migration which demonstrates an ability toinhibit spread of tumors.

Additional embodiments of the present disclosure comprise the use of thecompositions in vitro and/or in animal models humanized or otherwise topromote and/or assist in the regeneration of tissues, organs and bodyparts for use, but not limited to organ/tissue or body parttransplantation.

Examples of medical uses of the present disclosure include healing ofpathological wounds, such as through use of a contractile toroid forassisting the closure of slow healing wounds e.g., diabetic wounds asshown in FIG. 5 b.

Diabetic wounds are examples of difficult to heal wound can include, forexample, a wound that is often characterized by slower than normalre-epithelialization/closure inflammatory phase and delayed formationand remodeling of extracellular matrix.

The present disclosure can also assist in the healing of chronic woundsor wounds that do not heal. Wounds that have not healed within threemonths, for example, are said to be chronic. Chronic wounds include,diabetic, diabetic foot, ischemic, venous, venous stasis, arterial,pressure, vasculitic, infectious, decubitis, burn, trauma-induced,gangrenous and mixed ulcers.

Chronic wounds include, wounds that are characterized by and/or chronicinflammation, deficient and overprofuse granulation tissuedifferentiation and failure of re-epithelialization and wound closureand longer repair times.

Chronic wounds can include ocular ulcers, including corneal ulcers. Useof the disclosed invention in would healing and tissue regenerationwould include in humans and agricultural, sports and pet animals.

The present disclosure contemplates that the regenerative effects of thetissue engineered composition will include beneficial changes inmembrane excitability and ion transients of the heart, nervous system,uterus and other tissues in health and disease

There are many different types of arrhythmia that can lead to abnormalfunction in the human heart. All forms of arrhythmia have associatedmorbidity and can have the potential to result in sudden cardiac death(SCD). Tachyarrhythmias, like ventricular tachycardia and ventricularfibrillation are the predominant mechanisms leading to SCD. In theclinic, SCD is most commonly linked to coronary artery disease andsubsequent transient ischemia. These episodes of transient ischemia caninduce gap junction remodeling in un-injured tissues, and thisremodeling can then cause arrhythmia.

Common arrhythmias include bradycardias, tachycardias, automaticitydefects, reentrant arrhythmias, fibrillation, AV nodal arrhythmias,atrial arrhythmias and triggered beats. It is anticipated that the saidcomposition, in addition its use in regenerative restoration of heartstructures will be used to treat cardiac rhythm disturbances of thesetypes.

There are many diseases of congenital, genetic and acquired origins thatmanifest as a primarily electrical pathophysiology. In such casesaccompanying tissue injury is not a factor in the generation of thearrhythmogenic substrate. These include, but are not limited to, Long QTsyndrome, Short QT syndrome, Brugada syndrome, and several accessorypathway disorders. One example, Wolff-Parkinson-White syndrome (WPW) isa condition where an accessory bundle of muscle, expressing electricalconnection, links the atrium and the ventricle of the subject. Thisadditional pathway provides the substrate for a reentrant circuitbetween the atrium and the ventricle which when activated can result inventricular tachycardia, and potentially lead to SCD. The presentdisclosure contemplates that treatment of the subject with the tissueengineered composition will modulate the likelihood of this reentrantpathway to become activated. It is further contemplated that this effectwill be the result of the compositions modulation of membraneexcitability in the region of reentrant activity.

Arrhythmias can also be the result of molecular abnormalities in theworking myocardium. These molecular abnormalities can be caused by thecellular response to environmental stress, genetic mutations, infection,and other conditions. One example of this type of disease isHypertrophic Cardiomyopathy (HCM). HCM is the number one cause of suddencardiac death in patients under 30 years of age. This disease can betransmitted genetically and results in the unchecked growth of themyocardium without any signs of injury. It can be diagnosed with apreventative physical exam and/or thorough family history. In thiscondition, gap junction remodeling in the hypertrophic workingmyocardium leads to the increased incidence of arrhythmia and can causeSCD. This outcome is often seen as the otherwise healthy young personwho suddenly dies after a period of exercise. Examples of such subjectsoccasionally can be seen in media stories concerning young prominentathletes who die suddenly of an unexpected heart attack. The presentdisclosure contemplates that treatment with the provided compositionswill prevent the occurrence of unexpected arrhythmias in these subjects.

Other common arrhythmias include premature atrial Contractions,wandering Atrial pacemaker, Multifocal atrial tachycardia, Atrialflutter, Atrial fibrillation, Supraventricular tachycardia, AV nodalreentrant tachycardia is the most common cause of ParoxysmalSupra-ventricular Tachycardia, Junctional rhythm, Junctionaltachycardia, Premature junctional complex, Wolff-Parkinson-Whitesyndrome, Lown-Ganong-Levine syndrome, Premature VentricularContractions (PVC) sometimes called Ventricular Extra Beats, Acceleratedidioventricular rhythm, Monomorphic Ventricular tachycardia, Polymorphicventricular tachycardia, Ventricular fibrillation, First degree heartblock, which manifests as PR prolongation, Second degree heart block,Type 1 Second degree heart block, Type 2 Second degree heart block,Third degree heart block. It is anticipated that the compositions of thepresent disclosure can be used to treat cardiac rhythm disturbances ofthese types.

Common drugs used for arrhythmia treatments include class Ia drugs e.g.,Quinidine, Procainamide, Disopyramide, class Ib drugs e.g., Lidocaine,Phenyloin, Mexiletine, class Ic drugs e.g., Flecamide, Propafenone,Moricizine, class II drugs e.g., Propranolol, Esmolol, Timolol,Metoprolol and Atenolol, class III drugs e.g., Amiodarone, Sotalol,Ibutilide and Dofetilide, class IV drugs e.g., Verapamil, Diltiazem andclass V drugs e.g., Adenosine and Digoxin. The present disclosureanticipates that TGF-beta3, ACT1 or other components described herein inthe provided compositions can be used in conjunction with theseapproaches to treatment of arrhythmia.

Other arrhythmia treatments include: Anticoagulant therapies, electricaltreatments, electrical cautery, cryo-ablation, radio frequency ablation,implantable cardioverter-defibrillator, and implantable pacemaker. Thepresent disclosure anticipates that the compositions can be used inassociation with these approaches for the treatment of arrhythmia.

Epilepsy is a chronic neurological disorder characterized by recurrent,transient, unprovoked seizures, resulting from disturbed neuronalactivity in the brain. There is evidence that epilepsy is caused bydysregulated connexin coupling between neuronal cells and disturbancesto Cx43 have been noted in human hippocampus associated with severeepilepsy. Over 50 million people worldwide have epilepsy. Over 30% ofpeople with epilepsy do not respond to currently available medications.The uncontrolled electrical disturbance associated with epilepsy oftenleads to comparisons to cardiac arrhythmias.

Common forms of epilepsy include: Autosomal dominant nocturnal frontallobe epilepsy, Benign centrotemporal lobe epilepsy of childhood, Benignoccipital epilepsy of childhood, Catamenial epilepsy, Childhood absenceepilepsy, Dravet's syndrome, Frontal lobe epilepsy, Juvenile absenceepilepsy, Juvenile myoclonic epilepsy, Lennox-Gastaut syndrome, Primaryreading epilepsy, Progressive myoclonic epilepsies, Rasmussen'sencephalitis, Symptomatic localization-related epilepsies, Temporal lobeepilepsy, West syndrome. The present disclosure anticipates that thetissue engineered compositions described herein can be used to treatthese epilepsies.

The following medications are used for treatment of epilepsy:carbamazepine, clorazepate (Tranxene) clonazepam (Klonopin),ethosuximide (Zarontin), felbamate (Felbatol), fosphenyloin (Cerebyx),gabapentin (Neurontin), lamotrigine (Lamictal), levetiracetam (Keppra),oxcarbazepine (Trileptal), phenobarbital (Luminal), phenyloin(Dilantin), pregabalin (Lyrica), primidone (Mysoline), tiagabine(Gabitril), topiramate (Topamax), valproate semisodium (Depakote),valproic acid (Depakene), zonisamide (Zonegran), clobazam (Frisium) andvigabatrin (Sabril), retigabine, brivaracetam, and seletracetam,diazepam (Valium, Diastat) and lorazepam (Ativan), Paral, midazolam(Versed), and pentobarbital (Nembutal), acetazolamide (Diamox),progesterone, adrenocorticotropic hormone (ACTH, Acthar), variouscorticotropic steroid hormones (prednisone), or bromide. The presentdisclosure anticipates that the compositions described herein can beused in association with these drugs in treatment of epilepsy.

Other epilepsy treatments include: ketogenic diet, electricalstimulation, vagus nerve stimulation, responsive neurostimulator system(rns), deep brain stimulation, invasive or noninvasive surgery,avoidance therapy, warning systems, alternative or complementarymedicine. It is anticipated that the compositions can be used inassociation with these approaches to treatment of epilepsy.

The modulatory agents listed herein can be given in association with thepresent disclosure, such as described herein as a therapy or medicamentto improve the healing of wounds, injuries, disease processes,surgeries, congenital malformations and regenerating tissue in asubject. In summary, EMT-priming will be useful in all manner of medicaltreatments involving cellular therapy to synchronize the healing and/orregenerative capacity of engrafted cells.

The agent can be formulated with a pharmaceutically acceptable carrierto provide the desired final concentration for site-specific, transientor systemic effect in association with the present disclosure.

The modulating agent can be present in direct association with thetissue engineered device or present in a substantially isolated form; astate that will not change following mixing with carriers or diluents.

The modulatory agent can be administered integral with the tissueengineered construct. In this case the provided agent will be dissolvedin solution within the solution of the hydrogel or can be present asparticles, nano-particles or some other vector that releases the agentinto the healing tissue. The agent can also chemically bonded to themolecules of hydrogel itself. The hydrogel will contain ˜001% to about1.5% of active ingredient(s), about 2%-60% of active ingredient(s),˜2%-70% of active ingredient(s), or up to ˜90% of active ingredient(s).

The agent provided as part of the present disclosure can also bedelivered in a substantially isolated form independent of the tissueengineered composition. The route of delivery, compositions,preparations and medicaments of the present disclosure can be in gels,oils, foams, sprays, ointments, suspensions, instillations, salves,creams, solutions, emulsions, lotions, paints, sustained releaseformulations, or powders, and typically contain active concentrationsthat will be the same as those listed above for integral administration.

The modulating agents delivered as part of the present disclosure canalso be combined with a pharmaceutically acceptable carrier or diluentto provide a pharmaceutical composition. Suitable for this are isotonicsaline solutions, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition substances such as emulsifying agents,stabilizing or ph buffering agents can be present.

The factors used in association with the tissue engineered device can beadministered topically, orally, or parenterally. For example, thecompositions can be administered extracorporeally, intracranially,intravaginally, ophthalmically intraanally, rectally, subcutaneously,intradermally, intracardiac, intragastric, intravenously,intramuscularly, by intraperitoneal injection, transdermally,intranasally, or by inhalant. As used herein, “intracranialadministration” means the direct delivery of substances to the brainincluding, for example, intrathecal, intracisternal, intraventricular ortrans-sphenoidal delivery via catheter, needle or intravenous drip.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels (e.g., poloxamer gel), drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like can be necessary ordesirable. The disclosed compositions can be administered, for example,in a microfiber, polymer (e.g., collagen), nanosphere, aerosol, lotion,cream, fabric, plastic, tissue engineered scaffold, matrix material,tablet, implanted container, powder, oil, resin, wound dressing, bead,microbead, slow release bead, capsule, injectables, intravenous drips,pump device, silicone implants, or all bio-engineered materials.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders can be desirable.

Some of the compositions can potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Effective dosages and schedules for administering the compositions canbe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms disorder are effected. The dosage should not be solarge as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual doctor in the event ofany counter indications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products.

The dose effective provided with the tissue engineered compositions ofthe present disclosure for a given subject or wound can be determined byexperimenting via methods known to those skilled in the art or developedby experimentation by those skilled in the art using culture, animalmodels and other biomedical approaches. Therapeutically effective dosesare those that render therapeutic benefit in at least 50% of thepopulation, and that show minimal, low or no toxicity at the effectivedose. Other factors such as the route of administration, frequency ofadministration, and patient age, sex, weight, health, disease-profile,and other relevant medical information, the wound exhibited by thesubject and the modulating agent that is being used will be also used tocalculate effective dose. For example depending on the size of the woundtreated and scale of the tissue engineered composition dose can have tobe adjusted accordingly. Different active agents can be deliveredtogether or separately, and simultaneously or at different times withinthe day. The doses can be administered in single or divided applicationsand given at repeat intervals over a time course beneficial to thesubject or suitable for the therapeutic use at hand. Co-treatment orpre-treatment localizing co-factors (e.g., TGF-3B3 or ACT1) at the siteof injury (e.g., in a gel directly adherent to an MI) or disease can beparticularly effective alone or in combination with the providedinvention.

A suitable dose given with the tissue engineered composition can bebased on the mass of modulating agent per kg of body weight of thepatient, and include, from 0.00002 to about 200 mg/kg body weight e.g.,0.002 to about 50 mg/kg body weight. A suitable dose can however be from˜0.0002 to 0.2 mg/kg body weight e.g., 0.002 to about 0.060 mg/kg bodyweight. Doses from ˜1 to 100, 200, 300, 400, 500, 1000, 2000 microgramsper administration are appropriate. In certain embodiments, modulatingagent composition can be used at ˜0.0001 micromolar (μM) or 0.06 μM toabout 300 μM, or up to 500 μM or up to 1500 μM, or up to 3000 μM or upto 4200 μM or more, final concentration at the treatment site and/oradjacent to the treatment site, and any doses and dose ranges withinthese dose numbers.

The skin provides a model of scar reduction/regenerative healingeffects. However, the present disclosure contemplates toroidal or othermorphogenetically induced constructs being of use in the regenerativerepair and scar reduction in various tissues and organs including theskin, heart, brain, spinal cord and eye. The contemplated benefits wouldapply following injury, surgery, medication, chronic or acute disease,malformation and other normal or pathological processes causing loss oftissue structure and function, pathology and/or replacement with scartissue.

For instance, surgical repair of congenital structural defects can takeplace through use of contractile toroids in utero for assisting inclosure of defects in embryos such as ventricular septal defects of theheart. In regenerative medicine, the compositions and methods of thepresent disclosure can be utilized for a scaffold for tissue-engineeredcircularized muscular structures, such as synthetic venous valves orbladder sphincter valves. A further use in regenerative medicine is as abuilding block for tissue engineering complexes that are mechanicallyactive or responsive biological structures. Specific examples of thiscan include branched and unbranched tubular structures such as syntheticblood vessels (as shown in FIG. 5 b), aortic and pulmonary vessels,glandular ducts, lymphatic vessels, sinuses, lung bronchi andbronchioles, digestive tract, urethra, reproductive tubes and the like.Further examples can include the combination of regular and irregulartoroids to generate hollow organs or organ sub-structures such asbladders, gall bladders, gastrointestinal tract, pancreas lung air sacs,kidney nephrons and the like.

Previously described herein is the disclosure of novel compositionsgenerated in vitro (e.g., toroidal rings of cells). The induction ofthese compositions is via a novel method that recapitulatesembryonic-like morphogenetic processes in vitro similar to those thatoccur as a prelude to the generation of natural complexities of tissuestructure, function, and signaling in vivo. The novel compositions andmethods described herein represent transformative technologies in woundhealing, greatly enhancing the performance of a number of other woundhealing therapies. Current therapies based on stem cells usually involvethe introduction of dispersed cells into a diseased organ with littleregard for the synchronization and activation of the potentiality ofthese cells for regenerative repair. Here, evidence is provided thatthis present widely used approach of randomly introducing stem cells cannot be effective, perhaps even deleterious. Only when stem cells wereprimed in the culture dish using the present methods prior toengraftment into a wound was there a subsequent dramatic improvement inthe regenerative healing of the treated animal. The new principlesoutlined in this disclosure have the potential to revolutionize thestandard care for cell-based therapies of injuries or any therapeuticapproach in which stem cells are used as a therapy for patients.

The present disclosure can be better understood with reference to thefollowing examples.

EXAMPLES

In the present example, a novel method is described for harnessing EMTto generate repeatable and geometrically distinct microtissue units,including contractile rings and hollow spheroids. The standard protocolfor gel contraction involves premixing cells into a collagen suspension,allowing the collagen-cell mixture to polymerize as a 4 mm thick gel atthe bottom of a the circular well of a multi-well plate, detaching thegel after a period of culture and then measuring the amount that thecomposite contracts. Typical results for the contraction assay are shownin FIG. 1 a, where following detachment the gel is contracted by cellsshowing varying degrees of heterogeneous and mostly unorganizeddispersion in the circular three-dimensional matrix.

In a variation on this protocol, cells were added onto an alreadypolymerized gel and the gel was detached after 24 hours of culture.Following a subsequent 24 hours it was noted that quite unlike what wasobserved when cells were pre-mixed into the gel, cells addedpost-polymerization always formed into uniform circular rings ofrepeatable diameter (FIG. 1 b). It was also found that ring size can bepredictably altered by either undertaking the culture in a circular wellof smaller diameter (FIG. 1 c) or by addition to the culture fluid of3-30 ng TGF-b, a well known inducer of the EMT (FIG. 1 d). Confocalmicroscopy of rhodamine-phalloidin and nuclear-DAPI stained gelsindicated a two-phase structure with cells undergoing recruitment at theouter rim arranging in radial spoke-like arrays (FIG. 1 e), whereascells in on the inner rim organized into a contractile ring (FIG. 1 f).Biomechanical measurements undertaken on gels indicated that thetoroidal units efficiently exerted force on the gel that exceeded thatof pre-mixed gel-cell composites (mean, se, t-test). The resultsindicated that by actively forcing the cells to undergo a round of EMTduring invasion of the poymerized collagen gel, a morphogenetic eventhad been initialized that led to self-organization of the mesencyhmalcells into a contractile ring. This experiment was repeated with RECcells (Wada A M, Smith T K, Osler M E, Reese D E, Bader D M.Epicardial/Mesothelial cell line retains vasculogenic potential ofembryonic epicardium. Circ Res. 2003 Mar. 21; 92(5):525-31. Epub 2003Feb. 6, incorporated by reference herein), a rat embryonic stem cellline with similar effect. REC progenitor cells also organized into adistinct ring in the hydrogel when seeded onto the top of the gel andthe then detached. These higher order toroidal structures failed tomaterialize for all cell types tested including BMSCs and embryonic stemcells if the cells were passively mixed into a liquid gel and allowed toset in situ.

Further analysis revealed the result if the shape of the gel was varied.2 mm thick collagen gels were thus polymerized in square wells. As wasthe case with circular wells, when cells were pre-mixed into the gel,following detachment square gels contracted, but showed nothingdistinctive about the dispersion or organization of the gel-cellcomposite over a 48 hour time course (FIG. 4 b). However, square gels towhich cells had been added post-polymerization consistently formed intothree-dimensional spheroids of uniform shape and size with a singleapical pore after 48 hours of culture (FIG. 4 a). These spheroidal unitscan be filled with FITC dye via the pore and did not leak dye except atthe pore mouth, indicating that the spheroids were hollow and relativelywell sealed structures. Tracking over time suggested a step-wiseprocess, by which cells initially formed a star-like arrangement thatconcavely deformed the gel edges, then progressively folded the apicesof the square upwards to form the spheroid with its apical pore.

Formation of contractile rings or so-called actin purse strings is aprocess commonly found in biology (Martin A C, Kaschube M, Wieschaus EF. Pulsed contractions of an actin-myosin network drive apicalconstriction. Nature. 2008 Nov. 23; incorporated by reference herein).For example, contractile rings have been observed in neuropore closurein Drosophila and also elaborate to effect wound closure in mammalianembryos (Martin P, Lewis J. Actin cables and epidermal movement inembryonic wound healing. Nature. 1992 Nov. 12; 360(6400):179-83;incorporated by reference herein). Tissue-engineered contractile ringscan have uses in promoting wound closure (e.g., slow healing diabeticulcers) or in repair of congenital malformations such as closing septaldefects in the heart. A further potential application of self-organizedtoroids and spheroids is as microtissue units or building blocks formore complex biological structures, perhaps in combination with organprinting technologies. For example, it can be envisaged that contractiletoroids can be iteratively stacked to build a branched tubular structureresembling a blood vessel. In conclusion, the present example describesa method for generation of unitary microtissues based on the harnessingof self-organizing, morphogenetic processes. These microtissue units canprovide a basis for incorporation and elaboration of naturally occurringhistocomplexity and function found in living organisms into artificiallyengineered tissues.

Examples from Skin

In the next example, stem cells were primed using the method describedherein prior to engraftment into a wound. Adult bone marrow stromalcells (BMSC) were isolated from adult rat femurs and passaged andcultured to produce a pure population of BMSC. A small biopsy punch (8mm) was used to create a small, 8 mm diameter round wound on the back ofthe animal. The punch site was inlayed with the preformed collagen cellcontaining the BMSC cells (toroid) and/or peptide and two 4-0 prolenestitches were placed in the skin at the biopsy sight to hold the gel inplace. The collagen gel (1 mg/ml) was polymerized in a sterile hood andBMSC cells were treated with the ACT1 peptide (150 uM) and then addedeither on top of the 1.5 mm gel (toroid) or mixed into the polymerizinggel. Wounds were also treated with the gel only, gel plus ACT1 alone,gel plus cells alone and toroids with a control peptide. Animals wereallowed to heal for 30 days and then sacrificed and the pelts wereremoved and the wounds excised and surrounding skin were processed forstandard embedding in paraffin epidermal surface-up. From wound edge towound edge every 30th section was mounted on a glass slide and stainedwith H&E histochemistry. Images of the granulation in each section werethen recorded as single images or montages of 2-3 images. Generally15-30 serial 300 um-spaced sections were recorded per wound. Thegranulation tissue area, length of epidermal surface and number offollicles intersecting the epidermis were then counted or measured usingImage J software from each wound montage. Estimates of wound granulationtissue volume and the granulation tissue area measurements were recordedfor each section. Similarly, scar surface area was estimated as wasfollicle density in the scar epidermis. T-tests for paired samples werecarried using MS Excel (p<0.05). Measurements on treatments woundswithin individual rats were normalized to the gel only control wound asa baseline.

The ACT1-alone-treated wound had a scar that was smaller than thecontrols and most other treatments. However, the wound that receivedboth the BMSC toroid and ACT1 had a scar that was even smaller insurface area than the ACT1-alone-treated wound. This finding of improvedhealing for the combinatorial treatment over all othertreatments/controls was a consistent result. It was also noted thatthese same 2 wounds, Gel+ACT1 and Gel+BMSC Toroid+ACT1, showedconsistent significantly faster closure rates than the other 4 wounds.Qualitative appraisal of the wounds indicated the following pattern ofvariance in scar size: Gel+BMSC toroid+ACT1<(smaller than)Gel+ACT1<Gel+BMSC Toroid<Gel alone=Gel+BMSCs (non-toroidal)+ACT1wound=Gel+BMSC Toroid+Rev control wound. The same order of scar sizevariance in response to the treatment and control conditions was alsoobserved. Importantly, the combinatorial treatment of gels containingthe toroid of BMSCs and ACT1 consistently had the smallest scars at theend of the 30-day experiment.

The novel composition and method described herein represents atransformative technology in wound healing, greatly enhancing theperformance of a number of other wound healing therapies. Currenttherapies based on stem cells usually involve the introduction ofdispersed cells into a diseased organ with little regard for thesynchronization and activation of the potentiality of these cells forregenerative repair. Here, the present example provides evidence thatthis widely used approach of randomly introducing stem cells may not beeffective, perhaps even deleterious. Only when stem cells were primed inthe culture dish using the present method prior to engraftment into awound, was there seen a subsequent dramatic improvement in theregenerative healing of the treated animal. It is also that theactivation of cells within the gel can be undertaken in the subject insitu, following a brief priming period in which the cells are attachedto the gel in a culture dish. The present disclosure can be usedcoincident with the injury or can be introduced to a site on the subjectprior to a surgery or any anticipated tissue-disrupting procedure (e.g.,non-surgical dermabrasion) in order to precondition that site for thepurpose of scar reduction, improved tissue structure and function andregenerative repair. Constituents of the present disclosure (e.g.,EMT-primed cells, ACT1 and TGF-B3) can also be delivered alone ortogether for the purpose of preconditioning. The pre-conditioning can beat or repeated at 1, 2, 3, 4, 5, 6 hours or any other interval up to 24hours. The pre-conditioning can be at or repeated at 1, 2, 3, 4, 5, 6 orany other interval up to 48, 72, and 96 hours and up to a week.Following preconditioning and surgery further post-treatment with theinvention or constituents thereof can be undertaken. The data outlinedin the present disclosure have the potential to revolutionize thestandard care for cell-based therapies of injuries or any therapeuticapproach in which stem cells are used as a therapy for patients.

To estimate the amount of granulation tissue, scar surface area anddensity of follicles in scar epidermis, a serial histological sectioningthrough each healed 30-day wound was performed. Following fixation for 4hours in 4% paraformaldehyde, excised wounds and surrounding skin wereprocessed for standard embedding in paraffin epidermal surface-up. Theblocks were then serial sectioned at 10 um normal to the epidermalsurface. From wound edge to wound edge every 30th section was mounted ona glass slide and stained with H&E histochemistry. Images of thegranulation in each section were then recorded as single images ormontages of 2-3 images. Generally 15-30 serial 300 um-spaced sectionswere recorded per wound. The granulation tissue area, length ofepidermal surface and number of follicles intersecting the epidermiswere then counted or measured using ImageJ from each wound montage. Toobtain an estimate of wound granulation tissue volume the granulationtissue area measurements recorded for each section were entered into MSExcel and the number and spacing between sections were used in estimatesof scar volume. Similarly, scar surface area was estimated from thelinear measurements of epidermis from each section and number andspacing between sections. Follicle density in the scar epidermis wascalculated by dividing follicle counts by scar surface area.

T-tests for paired samples were carried using MS Excel (p<0.05).Measurements on treatments wounds within individual rats were normalizedto the gel only control wound as a baseline.

The following example further exhibits a treatment method using toroidsdramatically reduces scar tissue formation following wound healing. FIG.5 a illustrate a toroid prepared in a collagen gel as outlined in theearlier example. An experiment was undertaken using novel compositionsin a wound healing application (i.e., FIGS. 5 a and 6 b).

In these experiments, a circular collagen gel containing a toroid wasgenerated comprised of rat bone marrow-derived stem cells. The gel andthe BMSC toroid was then sutured into a circular 1 cm diameterexcisional wound on an adult rat (FIG. 8 b). The treated wound was donein association with 5 other control/treatment wounds on the samerat—i.e., there were a total of 6 equally sized 1 cm diameter wounds onthe rat—see FIG. 8 a.

From caudal (head) to tail and left hand to right hand the 6 wounds weretreated as follows (See FIG. 7):

1. The top (i.e., caudal) left hand wound on the rat dorsal mid-linereceived a circular collagen gel containing no cells or other treatment(Gel-only CONTROL). This wound was considered a non-treatment controland was expected to heal in a manner comparable to any wound of 1 cmdiameter on a healthy rat.

2. The top right hand wound received the collagen gel containing thetoroid of BMSCs, but no other treatment (Gel+BMSC Toroid treatmentwound). This treatment tested the effect of a toroid comprised of BMSCson wound healing.

3. The middle left hand wound received the collagen gel containing atoroid of BMSCs+ACT1 at 150 uM (Gel+BMSC Toroid+ACT1 treatment wound).This tested the effect of a combinatorial treatment of a toroidcomprised of BMSCs and ACT1 on wound healing.

4. The middle right hand wound received the collagen gel containing aninduced toroidal ring of BMSCs+reverse control peptide at 150 uM(Gel+BMSC Toroid+Rev control wound). This treatment controlled for theeffect of the Cx43-based peptide (i.e., ACT1) in a combinatorialtreatment.

5. The bottom (i.e., tail) left hand wound received the collagen gelcontaining ACT1 alone (i.e., no cells) at 150 uM (Gel+ACT1 treatmentwound). This treatment was a positive control, as it is alreadyestablished that ACT1 speeds wound healing and reduces scar tissue—inmouse and pig models.

6. The bottom right hand wound received the collagen gel containingBMSCs pre-mixed into the gel, but not activated into a toroidalring+ACT1 at 150 uM (Gel+BMSCs (non-toroidal)+ACT1 wound). Thisimportant control examined whether it was necessary for the stem cellsto be subject to the novel steps for inducing toroidal morphogenesis invitro, before being applied in combination with ACT1 to wounds.

The experiment was repeated on an additional 4 rats i.e., there were atotal of n=5 rats each receiving 6 wounds. The 5 rats were allowed toheal for 30 days, during which time photographs were taken of thehealing skin on each rat at set intervals. At the end of the 30 days,the wounded skin of each rat was sampled for histological analyses ofscar tissue.

FIGS. 8 and 9 are all from the same rat and are representative ofresults from the other rats.

In FIG. 8 a, the six equally sized 1 cm diameter wounds cut on thedorsal mid-line of a rat are shown prior to treatment on day 1 at thebeginning of the experiment (i.e., time 0). In FIG. 8 b, these samewounds are seen with each of the 6 gel treatments sewn in and secured by2 small sutures. FIG. 8 c shows the healed scars from each wound at theend of the 30 day experiment. The scar boundaries are outlined in red.Note from FIG. 8 c that the “Gel-only” control and the control wounds onthe right hand side of the rat had scars of similarly large dimensions.As expected the ACT1-alone-treated wound had a scar that was smallerthan the controls and most other treatments. However, the middle lefthand wound that received both the BMSC toroid and ACT 1 had a scar thatwas even smaller in surface area than the ACT1-alone-treated wound. Thisfinding of improved healing for the combinatorial treatment over allother treatments/controls was a consistent result in the 5 rats studied.

It is also noted that these same 2 wounds, Gel+ACT1 and Gel+BMSCToroid+ACT1, showed significantly faster closure rates than the other 4wounds. This result again was consistent between the 5 rats.

Serial histological sectioning was undertaken through the volume of eachhealed 30-day wound (i.e., on 20+ scars/wounds), three-dimensionalvolumes of wound granulation tissue were reconstructed (i.e., the scarprogenitor tissue) and the volume the volume and surface area of thegranulation tissue were carefully measured, as well as regeneration ofhair follicles in the epidermis overlying the healed wound.

FIG. 9 shows H&E histochemical stainings of single sections from themiddle of each of the six healed wounds from the same rat as shown inFIG. 8. These sections were one of the 10-20 serial sections taken perwound used for three-dimensional reconstruction of scar volume.Qualitative appraisal of FIG. 8 c indicates the following pattern ofvariance in scar size: Gel+BMSC Toroid+ACT1<(smaller than)Gel+ACT1<Gel+BMSC Toroid<Gel alone=Gel+BMSCs(non-toroidal)+ACT1wound=Gel+BMSC Toroid+Rev control wound. FIG. 9 confirms the same orderof scar size variance in response to the treatment and controlconditions. Importantly, the combinatorial treatment of gels containingthe toroid of BMSCs and ACT 1 consistently had the smallest scars of the6 wound conditions tested at the end of the 30-day experiment.

Referring to FIG. 10, a statistical analysis of the data fromquantitative wound histology was consistent with the qualitativeappraisal.

Gel+ACT1 and Gel+Stem Toroid+ACT1 had significantly smaller volumes ofgranulation tissue than the other 4 wound control/treament conditions(p<0.001). Moreover, the Gel+Stem Toroid+ACT1 treatment scars weresignificantly smaller in size/volume than wounds receiving Gel+ACT1alone (p<0.01). Quantification of scar surface area showed similarresults. Note also the increased density of hair follicles in thetoroid+ACT1 compared to the other control wounds. For example, comparedto the Gel-only control, the combinatorial toroid+ACT 1 treatmentprovided a highly significant (p<0.00001) 275% increase in follicledensity in the wound epidermis. This result demonstrates that the scarreduction prompted by the combinatorial treatment was accompanied by astriking regeneration of normal skin histoarchitecture.

An interesting feature of the quantitative analysis was that thecombinatorial treatment of Gel+Stem Cells (non-toroidal)+ACT1 wounds didno better than the Gel alone control. Indeed, in some rats it appearedthat this combination did worst of all compared to the other conditionsapplied to wounds. This result indicates that in the context of theexperiment, BMSCs must be morphogenetically transformed into theembryonal-like composition for the combinatorial effect to occur.Moreover, the results indicate that stem cells that are not composed inthe provided composition, even the presence of ACT1, are not sufficientto provide benefit during wound healing. These results indicate that thecombinatorial effect of the provided composition with ACT1 will benefitto wound healing and scar reduction over and above each of these twofactors alone.

The toroid effects are likely contributed to by signaling factors fromits constituent cells, in addition to biomechanical forces, genetic andepigenetic influences exacted by the provided composition. As such,conditioned media or factors secreted by BMSCs when organized in thecomposition contribute to the combinatorial effect. As outlinedelsewhere in the present disclosure, this combinatorial effect will beobserved in conditions that include delivery with the providedcompositions, cell-free extracts from the compositions, extractcomponents and/or conditioned media from the provided compositions.

The results outlined herein indicate that the presence of cells alone inthe wound were not sufficient to promote regenerative healing. The cellsneed to be prompted to undergo the morphogenetic transformation inducedby the steps described herein for optimal scar reduction/regenerativehealing effects.

In still another example, FIG. 11 illustrates three healed 31 day-oldscars from full-thickness 1 cm circular excisional wounds performed onthe dorsal mid line of the same adult rat. All 3 wounds were generatedon the rat on day 1 of the 31 day healing time course within the same 30minute period of surgery. Healed scars were exposed by shaving the ratand depilating with Nair™ on day 31. Scar is delineated by a relativelack of hair follicles and variable skin tone-generally lighter and/ormore heterogeneous than surrounding skin.

FIG. 11 A-C show the scars without annotation. A′-C′ show the same scarareas on each healed wound outlined in black.

A) Wound immediately received the toroidal ring of bone marrow stemcells (BMSCs) in gel (BMSC+Gel) following the surgery on day 1 of theexperiment. As shown elsewhere in 4 other rats, this treatment conditionresulted in a scar at ˜30 days whose superficial appearance did notdiffer significantly from control wounds receiving only collagen gel.The scar in A thus serves as a comparative control for the other twoscars shown in the FIG. 11 (i.e., B and C).

B) Injury received 3 ng/ml TGF-B3 in a collagen gel immediately(TGF-Beta3+Gel) following surgery on day 1 of the experiment. TGF-B3treatment caused a 19% reduction in the superficial area of visiblescar.

C) Injury received the a toroidal ring of activated BMSCs together with3 ng/ml TGF-B3 in the collagen gel (BMSC+TGF-Beta3+Gel) immediatelyfollowing surgery on day 1 of the experiment. Similar to data in thisdisclosure showing that a combinatorial treatment of a toroid of BMSCswith ACT1 causes striking reduction in scar area, it was determined thata combinatorial treatment of a BMSC toroid and 3 ng/ml TGF-B3 results ina 64% reduction in scar size compared to the wound receiving the BMSCtoroid control (A-A′) and a 55% decrease in scar size compared to thewound treated with TGF-B3 alone. Note that the tone and uniformity toneof the scar in C matches the surrounding skin more closely than thatseen in the scars in A and B. These trends are confirmed in thehistological analyses in FIG. 12.

Based on these data it is concluded that activated BMSCs in the gelconstructs described herein combine with compounds including ACT1 andTGF-B3 to promote scar-free healing in an embryonal-like manner. Thus,either ACT1 or TGF-B3 when provided acutely alone reduces apparent scararea. However, when either is given in combination with a BMSC toroid,both ACT1 and TGF-B3 prompt even greater reductions in scar sizecompared to controls and to when ACT1 and TGF-B3 are used alone. ACT1and TGF-B3 are contemplated to be 2 members of a broad class of factorsas described further herein that combine with cells activated asprovided in the present disclosure to promote scarless, embryonal-likehealing in an adult tissue. As was the case with the combinatorialtreatment of ACT1 and cells mixed into the gel, when TGFb3 was givenwith BMSCs mixed into the gel the benefits on scar reduction andimproved regenerative healing seen with the TGF-B3 and the presentdisclosure were not observed.

Examples from Heart

A therapy capable of regenerating cardiac muscle lost by injury ordisease has not yet been convincingly demonstrated. Excess formation ofscar tissue is the normal response to injury or disease in the humanheart, not regeneration. Tipping the balance from the normal scarringresponse to regeneration in the heart has thus far proved difficult, ifnot impossible. The provided compositions will be provide a means ofregenerative repair of the heart following disease (e.g., heart attack),surgery, injury of congenital malformation.

One of the commonest injuries to the heart is a myocardial infarction(MI) that occurs as a sequalae to coronary heart disease (CHD). CHD isthe biggest killer of people in developed countries. During an MI or“heart attack” there is a sudden failure of coronary circulation. If thepatient survives, the MI scar may cause sickness or death from loss ofcardiac function (heart failure) or prompt the development oflife-threatening arrhythmias. The “EMT-primed” cells in the providedinvention would be deployed to reduce scarring following MI and thusameliorating morbidity and mortality associated with CHD.

A new method has been developed for injuring the heart in an animalmodel that was specifically designed to increase the ability todetermine whether the therapeutic approach causes regeneration ratherthan the normal process of formation of scar tissue following an injurysuch as MI (FIG. 13 and O'Quinn et al. Circulation, 118: 495, 2009,incorporated by reference herein). This method involved delivering afreezing injury to the heart that always generated a non-transmuralwound of consistent size and depth in the left ventricular wall muscle.Because wound size was consistent between mice, the exact amount of scartissue that would be deposited in the heart in each animal injured canbe ascertained. More importantly, the consistency of the lesion enabledthe determination with certainty that has not been previously achievableby others as whether newly regenerated muscle was present in the healedinjury. Example of the usefulness of this injury method in detectingcontrol and treatment effects on regeneration of myocardial structureand function is provided below.

To undertake the novel injury model, 12-24 wk female CD1 mice (CharlesRiver) were used. Mice were anesthetized (isoflurane), intubated and aleft thoracotomy was performed at the 4th intercostal space. The LV wallwas cryo-injured by exposure for 5-sec to a liquid-N₂ chilled 3 mmcircular flat-tip probe (Brymill: CRY-AC-3) such that the LV surface wasslightly depressed. In the case of treatment of the animal model withthe composition cryo-injury, the mouse receives EMT-primed BMSCs in geltogether with 3 ng/ml of TGF-beta3 over the cryo-injury, and the gel isthen held by 2 small dissolving sutures on the surface of theepicardium. Cel-Tak™ adhesive (BD Biosciences(11)) or other surgicaladhesive can also be used to secure the gel to the wound. Surgicalwounds are then closed using 6-0 silk sutures (Ethicon) and sealed withNexabond™.

In another example of an MI injury adult (12-24 wk) CD1 mice are used.Mice are anesthetized and a left thoracotomy performed at the 4thintercostal space to expose the heart. A suture is then placed throughventricles mid-way down the anterior interventricular branch of leftcoronary artery and great cardiac vein. The suture is tied, blocking offperfusion of the ventricle below the level of the occluded arterycausing an MI-like mouse model of a human “heart attack”. Cel-Tak™adhesive (BD Biosciences(11)) is then used to secure treatment gelscontaining EMT-primed BMSCs together with 150 uM ACT over thecryo-injury.

The gels containing toroidal rings of EMT-primed mouse BMSCs aregenerated as follows. Bone marrow is collected from the femurs andtibiae of adult (12 week old) CD1 mice, pooled and CD45+ mononuclearcells isolated by gradient separation and FACS-sorting. Followingisolation, dispersed BSMCs are added to the top of pre-polymerizedcollagen gels in 6-well microplates and the cells allowed to attach over24 hours in a fortified media that supports BSMC survival. The gel/cellsare then detached from well walls and the free floating gels culturedfor a further 24 hours, over which time the characteristic toroids formin the gel.

Mouse iPS cells are generated from murine skin fibroblasts bytransduction with OCT4, SOX2, c-MYC, and KLF4 carried in a retrovirus asdescribed previously (Li et al. J Cell Biochem. 2009; Takahashi andYamanaka. Cell. 2006; 126(4):663-676, incorporated by reference herein).The iPS cells generated from mouse skin fibroblasts will be maintainedon irradiated MEFs feeder layers in standard ES medium. Validation ofreprogrammed state of murine iPS cells will be done using antibodies tonuclear marker Nanog, and the surface markers, TRA-1-60 and Ssea4.Following transduction with the reprogramming retrovirus reprogrammedcells will be tested for stemness as evidenced by their having achievedsignificant milestones including 1) proviral silencing (i.e., silencingof viral vector encoded GFP) and 2) induction of endogenous Nanogexpression. With further culture these GFPdim/−, Nanog+ cells go on toexpress TRA-1-60 as described by (Chan et al. Nat. Biotechnol. 2009;27(11):1033-1037, incorporated by reference herein). One iPS cells areconfirmed the cells will be EMT-primed in gels as described in thepreceding paragraph for BMSCs.

Following surgery and application of the gel and EMT-primed BMSCs (orEMT-primed iPS cells) to either freezing or ligation injuries of theheart, mice are ventilated until respiring spontaneously, warmed andgiven analgesia until recovery. Following surgery the mice are measuredweekly for heart function using M-mode echocardiography for a 6-12 weekperiod. Some hearts are also tested for propensity to developarrhythmias using an induced arrhythmia protocol and electricalactivation mapping with voltage sensitive dye as has been describedpreviously (O'Quinn et al. Circulation, 118: 495, 2009, incorporated byreference herein) At the end of the said period the mice are sacrificedand the healed cryo-injured hearts are assessed using standardhistological and immunohistological approaches for scar tissue formationand regeneration of new cardiac muscle in place of what would normallybe scar in a untreated control animal.

Using the said cryo-injury model in a mouse model, it has been shownwith confidence (i.e., p<0.05), for example, that release of ACT1 from amethyl-cellulose patch directly on the injury results in significantimprovement in LV diastolic and systolic function over a 8 week timecourse (n=21 animals). This improvement in mechanical function wasassociated with significantly increased scar uniformity. Treated heartsalso showed higher and more uniform Cx43 in myocytes bordering the scar,as well as elevated levels of phosphorylated s368 (ps368) Cx43 from asearly as 2 hours following injury. ps368 upregulation is associated withischemic preconditioning and this elevation of ps368 contributes to thebeneficial effects of ACT1 following myocardial injury. It is alsoexpected that delivery of ACT1 or other agent (e.g., TGF-B3) in apre-conditioning period prior to injury will have a beneficial effectand that this effect will be improved still further by the furtherdelivery of the provided invention. Consistent with evidence thatdownregulated and disordered Cx43 at the infarct border zone is a keyfactor in cardiac conduction disturbance, it has been determined thatthere was a dramatically reduced (p<0.05) frequency and severity ofarrhythmias in ACT1-treated animals as assessed by electrophysiologicalstudies (pacing and S1-S2 protocols) (n=22 animals) (O'Quinn et al.Circulation, 118: 495, 2009).

In another example of the novel method, analysis of heart pump functionby echocardiography showed that one week following injury in a secondgroup of treatment mice (mice in which bone marrow containing stem cellswas infected with an shRNA lentivirus) and control mice (i.e., micesimilarly receiving a control virus) showed a similar (˜20%) decline inthe efficiency of heart pumping function—as measured by % ejectionfraction from the left ventricle. Ejection fraction is a standardclinical measure of cardiac pumping efficiency. This decline indicatedthat just after freeze wounding both treatment and control hearts hadreceived a similar initial degree of injury as reflected by theirsimilar reduction in function over the first week. However, at the endof the following 4 weeks, a stage at which it would be expected that thehealing of the injury to the heart and scar formation to be nearingcompletion, cardiac pump function of the treatment had improved to be<98% better than that of controls. Remarkably, by 4 weeks heart pumpfunction in the treatment had recovered to levels identical to those ofa normal uninjured heart. Meanwhile in controls, pump function haddeclined at the 4 week period by 50% compared to uninjured hearts.

The improvement in % fractional shortening of the left ventricle isanother clinically used measurement of cardiac function andcontractility. Percent fractional shortening improved by more than 120%in the treatment relative to control at 4 weeks following injury. As wasthe case with ejection fraction, treatment caused a recovery of %fractional shortening levels to those of a normal, uninjured heart at 4weeks, whereas controls continued to show significant declines in thisindex of cardiac contractile function.

The systolic and diastolic volume of the left ventricle during thecardiac contraction cycle are two other commonly used indices of cardiacfunction. Increases in these indices are recognized as indicative of aloss of cardiac function and are viewed by clinicians as disease markersfor the development of eventual heart dilation, heart failure and death.The diastolic volume of the left ventricle of treatment wassignificantly improved, being 40% less dilated than that of control.More remarkably, left ventricular systolic dimension was improved tobe >75% lower than controls. Putting this another way, at 46.5, the leftventricular volume of control at systole was 5-times more dilated atsystole than that of the 10.61 value measured from the echocardiogramsof treatment. Treatment also caused both left ventricular volume indicesto recover to levels found in the normal, uninjured heart. No suchrecovery to normality has ever been noted to occur in controls.

The data at 4 weeks post-injury led to the conclusion that the mice thathad received standardized cardiac injury and treatment unexpectedlyrecovered to normal cardiac pump function and contractility. In furthercontrast to controls, there was no sign of pathological cardiac dilationindicating that treated hearts were progressing to heart failure andeventual death.

Echocardiographic measurements of % ejection fraction, % fractionalshortening, and left ventricular volume at diastole and systole wererepeated at 6 weeks. These measurements indicated that the improvementin these parameters found at 4 weeks were sustained 6 weeks followingtreatment and injury. By contrast, none of these cardiac functionparameters showed any improvement in the control at 6 weeks and were formost part were similar to the depressed measurements taken in controlsat 4 weeks. Indeed, left ventricular volume at diastole showed furthersignificant deterioration in the control indicating a continuingprogression toward heart failure in the untreated control.

Second, the unexpectedly large beneficial effects on regeneration ofcardiac muscle and reduction of scar in the injured heart were noted.Following echocardiography at 6 weeks, hearts were removed formorphological and histological analyses. A large pale scar was evidenton control hearts with no sign of regeneration. This large scar extendednearly to fully incorporate the boundaries of the initial injury. Bycontrast, the area of initial injury in a treated heart showed only aminimal amount of visible scar at the 6 week time point. In quantitativeterms, less than 10% of the initially injured area on the control heartis cardiac muscle. By contrast, the treated heart showed a 70-90%regeneration of normal cardiac muscle. Thus, in summary the unexpectedability to prompt a full recovery of function in treated hearts iscorrelated with an equally impressive and unexpectedly extensiveregeneration of normal cardiac muscle at the injury.

That regenerated muscle was present was further confirmed by histologyof the hearts. Myocytes in treated hearts were found throughout the scarwith a particular concentration of these cells near the epicardialborder of the scar. This sub-epicardial population was notable for anumber of reasons. First, it is direct evidence for myocardialregeneration. The freeze injury is via a liquid nitrogen-cooled probeapplied to the outer surface of the heart generating a hemi-sphericalinjury volume. During the freeze injury, the broadest sector of lethallyfrozen tissue is at the epicardium just under the freezing probe, i.e.,the site where the “new myocytes” can be seen after 4 weeks of healing.Thus, this zone of sub-epicardial “new myocytes” must have regeneratedover old necrotic tissue frozen near the epicardium—the previous cellsat this location could not have survived the freeze injury. Indeed, inmore than 20 control hearts subject to standardized freeze injuryevidence of regeneration at the sub-epicardium was never seen. Second,the myocytes in this sub-epicardial zone were compact and highlyaligned. This means that the treatment methods of the present disclosurehad not only induced “new myocytes”, it had also the regenerated theprecise tissue organization that existed at this locale in the heartprior to injury. Thus the treatment had unexpectedly regeneratedstructure at both cellular and tissue scales—i.e., in addition torestoring function at the organ level. Thirdly, it is noted that these“new myocytes” are contiguous with adjacent myocardium. Cx43immunolabeling indicates that these new myocytes also express the gapjunction protein. Such tissue organization is consistent withelectromechanical integration with surrounding myocardial tissues andthe lowering the likelihood of arrhythmia. As noted previously, it iscontemplated that the novel composition of the present disclosure willprevent arrhythmias.

It can also be noted that the collagen staining appears significantlypaler in the treated hearts indicating that collagen organization isdifferent from that of controls. Whereas much cardiac research isfocused on attempting to promote adult myocyte cell cycle re-entry toregenerate cardiac muscle, the novel approach described herein leads tomodification of scar organization in vivo. It is posited that the scarin the treated animals is a “better scar,” permitting a new type ofremodeling of this region with new myocytes. Finally, the sectionreveals that the extent of scar tissue as indicated by comparing thearea of scar tissue is significantly less (>60-70% less) in thetreatment compared to controls. This means that the treatments of thepresent disclosure can have an unexpectedly profound effect of tippingthe balance between scar formation, organization and inducing amultiscalar regeneration of functional myocardium in the injured heart.

In a further example in heart, the provided composition can beintroduced via keyhole surgery in a human subject who has suffered an MI(i.e., preferably within 1 week of the MI) under full anesthetic by asurgeon into the minimally disrupted pericardial sac of the subject viaa catheter. The stem cells activated (i.e., EMT-primed) by the methoddescribed herein in the composition are derived from a tissue-matchednon-autologous source or taken from the subject themselves. For example,from previously sampled and stored bone marrow stem cells biopsied fromthe patients hip or peripheral blood or iPS cells generated from thesubjects own skin fibroblasts according to standard protocol. Thesurgical wound in the patient's skin is repaired by a small suture andthe patient allowed to recover.

In another example, the compositions would be sutured or secured bysterile surgical adhesive into place over an acutely healing MI whilethe subjects heart is exposed during coronary artery bypass graftsurgery (CABG) and the like. Following CABG surgery the healing of themyocardium of the subject would be monitored for improvement in cardiacfunction by routine EKG, ambulatory EKG, echocardiography, blood assaysand other tests of cardiac well being and healing that a qualifiedclinician deemed necessary for the recovery of the subject.

Examples from Spinal Cord

Subjects with acute spinal cord injuries to the central nervous system(CNS) represent a seriously problematic group for whom even a smallneurological recovery of function can have a major influence on theirsubsequent independence. The provided compositions would be especiallyuseful in patients with a complete cord injury who normally have a verylow chance of recovery. For optimal recovery of function the compositionwould optimally be applied acutely or sub-acutely within 1 week of theinitial injury. The prognosis of incomplete cord syndromes would also beimproved by the composition.

In one example, a subject with an acute anterior cord injury due to aflexion injury of the cervical spine would have surgery performed toexpose the dorsal aspect of spinal cord at the level of the injury. Agel containing a toroidal ring composed of EMT-primed stem cells asdescribed herein is then placed directly on the injury. Single ormultiple compositions are applied depending on severity of the injury.The surgical wound exposing the spinal cord injury is then sutured shut,enclosing the composition in situ. The stem cells activated by thepresent method are derived from a tissue-matched non-autologous sourceor taken from the subject themselves (for example, from previouslysampled and stored bone marrow stem cells or iPS cells generated fromthe subjects own fibroblasts). Improvement in function is assessed by adoctor at intervals (e.g., 6, 12, 26 and 52 weeks) following treatmentby neurological outcome tests including assessments designed to measuremotor activity, pinprick skin sensitivity and recovery of sensation.CT/MRI of the spine at the level of injury is also undertaken to monitorthe healing progression of the subject. Medium- and long-term managementwould then be directed towards rehabilitation, including physiotherapyand occupational therapy to enable as full recovery of function as ispossible following the treatment.

In one aspect the recovery of spinal function will occur because ofregeneration of new spinal cord neural connections from stem cells. Thisreparative aspect will occur in other CNS and PNS (peripheral nervoussystem) tissues. In another aspect, the recovery of spinal cord functionwill be contributed to by reduction in inflammation, swelling, odema andtissue loss associated with placement of the composition. Assay of thiscan be tested in animal models. For example, following injury to ratspinal cord in vivo, rats are treated with the composition. Solublefluorescein-isothionate-tagged BSA (bovine serum albumin) or Evans bluedye is then injected into the tail vein. Control animals show leakage ofthe dye from the vascular system into tissues within and surrounding thespinal cord. However, animals treated with the composition demonstrateonly limited dye leakage, with it majorly being confined with intactvascular structures. In the case of the CNS tissues such as the brainand spinal cord, this is due to the composition promoting themaintenance of the blood-brain barrier. However, the maintenance ofbarrier function should in some aspect be seen in all tissues of thebody. The results will thus indicate that leakage of thecapillary-vascular system is not restricted to the CNS (e.g., spinalcord, brain, retina) and that a broader range of medical applications,such as for treatment of conditions of blood vessels, would benefit fromtreatment with the provided composition.

Spinal cord experiments are carried out on adult SD rats as previouslydescribed by Banik and co-workers (Sribnick et al. J Neurosci Res. 2006October; 84(5):1064-75, incorporated by reference herein). Rats areanesthetized and laminectomies are performed at T-12. Trauma isadministered by dropping a weight of 5 g from a height of 8 cm onto animpounder (0.3 cm in diameter; 40 g·cm force) gently placed on thespinal cord. Treatment gels (i.e., 5 mm diameter including EMT-primedstem cells) and controls (as per eye and heart injury) are immediatelyapplied and wounds sutured closed. Spinal cord edema is assessed at 48hrs post-injury, as described above. Cell death caused by compressioninjury is also assessed acutely on 5 μm sections of spinal cord from thelesion, which are co-labeled with NeuN and TUNEL staining as a markerfor neurons and cell death respectively. Assessment of inflammatory cellinfiltration (e.g., microglia and macrophages) will be done using OX42and ED2 antibodies. To determine the long-term benefits of treatment oftreatment with EMT-primed BMSCs; the functional and behavioral recoveryof rats are tracked over time courses up 6 months following injury andNeuN and GFAP immunohistology will be used to assess glial scar andneurogenesis across the scar.

Examples from Eye

Normal eyesight is dependent on the transparency and regular curvatureof the cornea. The histoarchitecture of the cornea is similar to that ofskin—consisting of a stratified epithelium overlying a collagen-richstromal matrix embedded with fibroblastic cells (e.g., keratocytes),although is largely avascular except at the periphery. Severe injury,surgery (Corneal refractive surgeries (CRS) such as photorefractivekeratectomy (PRK)) and certain disease processes can lead to the loss ofcorneal transparency via activation of fibrotic/scarring processes inthe corneal stroma. The resultant severe fibrosis of the cornea isdifficult to treat and typically requires corneal transplant, which maylead to further complications. A safe and effective approach to reducingcorneal scarring complication such as provided by the presentcomposition would thus be welcomed by ophthalmologists and eye surgeonsalike.

Minor scratches on the cornea are common and the composition is notenvisaged to be used for normally healing minor injuries. However, thecomposition would be of use in the treatment of more serious injuries tothe cornea that may occur from small flying particles when drilling,sawing, chiselling, grinding, lawn mowing, and so on without eyeprotection and also from chemical burns such as that resulting formcaustic solutions, acids, wet concrete and the like. Also thecomposition would be used in patients receiving CRS/PRK surgeries thatmay present high risk profiles such as those displaying wide pupils orevidence of poor wound healing such as might occur in a diabeticpatient.

Following standard sub-acute stabilization and cleansing by a clinician,a subject suffering a severe chemical burn would have a collagen gelcontaining a toroidal ring composed of stem cells and 180 uM ACT1prepared as described herein, placed directly on the injury. Preferablythe treatment would undertaken within 1 week of the initial injury.Single or multiple compositions can be applied depending on severity ofthe injury. Antibiotic eye drops would then be placed in the eye toprevent infection. The composition can also be place with a associationmembrane to further aid healing. The eyelid would then be temporarilysutured closed, to retain the composition and a bandage would then beplaced over the closed eye. Painkillers such as paracetamol or ibuprofenwould be used to ease pain over the subsequent healing process. 7-14days later the lids would be released and repair of the cornea assessedby an ophthalmologist for inflammation, scarring and other clinicalindications of corneal healing. The stem cells activated by the methoddescribed herein in the composition would be derived from atissue-matched non-autologous source or taken from the subjectthemselves (for example, from previously sampled and stored bone marrowstem cells or iPS cells generated from the subjects own skinfibroblasts). Improvement in function are assessed by a doctor atintervals (e.g., 6, 12, 26 and 52 weeks) following treatment by visiontests. An eye patch to cover the eye would not normally be advised after10-14 days following injury as this may impair the healing process.

An animal model of corneal injury has already been published (FIG. 14and Chen et al. Invest. Ophthalmol. Vis. Sci. 2009; 50: 2480,incorporated by reference herein). In this model, adult (12 wk) SD ratsare anesthetized and the central cornea treated with 20% ethanol for 30seconds using a 3-mm marker placed on the corneal surface. The cornea isthen thoroughly rinsed with saline and the loosened epithelial layerremoved using a detaching spatula. A treatment (i.e., gel withEMT-primed non-autologous BMSCs) or control gel is then placed in thealcohol burn injury and the eye-lid sutured shut for 48 hours to holdthe gel in place.

Corneal wound closure is determined by administering 0.25% fluoresceinsodium eye drops and digitally capturing the cornea under a fluorescentstereomicroscope at 0, 48, 72, 96, and 120 (closure is usually completeby 120 hours in rat) hours post-injury (e.g., FIG. 14). Levels of scartissue deposition and transparency is assessed on whole mounts ofisolated corneas 30 days post injury. Corneal tissue are also be subjectto standard histological and immunohistochemical studies on tissuessections to assess corneal epithelial and endothelial integrity andcollagen organization and myofibroblast (alpha-SMA) density in thestroma.

Example from Tissue Engineering

Loss of skeletal muscle mass is an important problem for surgeons.Skeletal muscle has some ability to regenerate from endogenous stemcells called satellite cells. However, if the injury is large, thisnatural reparative ability can be overwhelmed. In such cases, muscle isnot regenerated and scar tissue replaces lost muscle—if the patient isfortunate.

One clinically important example of injuries involving muscle that canbe difficult to repair are ventral hernias (also known as incisionalhernias). Annually, over 2 million abdominal operations are performed inthe United States. (Millikan K W. Surg Clin N Am 2003; 83(5):1223).Given a failure rate for abdominal closures of 11 to 20 percent, it isnot surprising that over 100,000 ventral hernia repairs are attemptedeach year in the United States alone. The incidence of ventral herniashas remained relatively stable over the last 75 years despite manymedical advances.

The repair of ventral hernias typically involves the closing the herniawith a synthetic mesh or more recently decellularized human dermis(Alloderm, LifeCell). Although these methods effectively “patch thehole” they lack the ability to reconstitute the lost abdominal muscle.The mesh imparts no contractile function and with large hernias it isineffective at producing counter pressure from the contracture ofremaining abdominal musculature. These repair techniques do little toreestablish the dynamic role of the abdominal wall in support of thetorso and lumbar spine. With adynamic repairs, force vector summation ofabdominal wall contraction is focused on the repair itself Mesh repairsare also associated with bowel obstruction (5%), enterocutaneousfistulae (2-5%), and infection (1-2%). The aggregate incidence of longterm complications associated with mesh repair approaches 27% (Mudge andHughes L E. Br J Surg 1985; 72:70-1). The following example illustrateshow the present disclosure can be used to repair an experimental ventralhernia in a rat—by extension in a human subject.

To create the ventral hernia model, 250 gram male Sprague Dawley ratsare used. This size male rat has sufficient tissue for isolation ofsatellite cells, creation of the abdominal defect and has maturedsufficiently to be considered adult in phenotype. After generalanesthesia is achieved, the animal is prepped in standard surgicalmanner. A 1 cm×1 cm excisional wound is then generated in the abdominalmuscle through to the cavity of the abdomen. To isolate autologoussatellite cells from skeletal muscle of the same rat, a muscle biopsy(0.5 mm×0.5 mm×0.2 mm=05 cm³) is extracted from the vastus lateralis andplaced in mosconas on ice. This provides the 10 to 1 expansion of cellsrequired to repair the defect, The biopsy wound is approximated andclosed by suture. The sampled muscle tissue is rinsed vigorously withPBS at least three times to remove blood. The tissue is then mincedthoroughly with scissors to dislodge adherent fat and washed severaltimes with cold PBS. Warmed and gassed protease is added (sigma #P-5147;1.25 mg/ml in Krebs Ringer Bicarb. Buffer (Cat #K4002)) to the tube withthe tissue at a concentration of 1:5 (enzyme:tissue), followed by 1.25hours shaking incubation at 37° C. The tube is centrifuged and thepellet is resuspended in 25-30 ml of high serum media (DMEM+25% FetalBovine Serum+1% Pen/Strep antibiotic+0.1% Gentamycin). DNAse is addedand the tube is shaken vigorously and centrifuged to collect the sample.Spun supernatants are then panned onto 150 mm dishes with 25-30 ml mediafor 1.5 hours at 37° C. in the incubator. The cells are dislodged with0.25% trypsin-EDTA when cells are at least 90% confluent, counted andseed onto CtCs. A sister culture of satellite cells is then created incollagen coated culture dishes. The cells are then characterized byimmunolabeling for Pax 7, Myf5, MyoD, and sarcomeric myosin (MF20). Inprevious studies, the satellite cell cultures are 80+% positive for Pax7and MyoD.

For generation of EMT-primed skeletal muscle stem cells, 30-50 collagengels are prepared in 2 cm diameter circular wells as described above.Dispersed satellite cells (12×10⁶ per well) are then applied to thewell. The cells are allowed to attach and culture of the collagensubstrate for 24 hours and then the gel is released as per standardpractice for the present disclosure. Alternately, the gels can bereleased after cell attachment is achieved, static or dynamic strain isthen applied to generate preferred alignment and differentiationpotential of the adherent cells. The gels can also be soaked in ACT1,TGF-B3 or other compound assisting muscle regeneration by the stemcells.

Following a 24 hour period in culture, circular gels containingEMT-primed stem cells can then be stacked within a single well, eachlayer being adhered to the next by small dab of Cell-Tak™ at the geledge. The cylindrical 3d assembly of gel layers with EMT-primed skeletalmuscle cells then has a suture threaded through the middle of its longaxis, removed from the culture well and then placed in the openexcisional wound in the abdominal muscle of the rat. The suture threadthrough the cylinder of EMT-primed stem cells stabilizes the assemblyand also is used to secure it in place. To increase the robustness ofthe repair multiple 3d tissue engineered constructs of EMT-primedsatellite cells can be applied to the ventral hernia. The repair site isthen covered with an appropriate surgical membrane and wound dressing toprotect the wound and implanted tissue engineered device. Animals arethen sampled at time points between initial wounding and 16 weeks.

In the rat model, inflammatory response, scarring and skeletal muscleregeneration can be assessed using histochemistry andimmunohistochemistry (e.g., Pax7, MyoD, MF20 expression) of the repairedabdominal tissues using standard approaches. Functional assessment oflive tissue from the repair can be done by taking regenerated musclefrom the repair placing in a muscle bath, oxygenated (95% O2&5% C02)Krebs solution maintained at 37° C. at pH 7.4, and undertakingphysiological tests of muscle function: isometric contraction,length/tension relationship determination, and breaking stress andstrain. In human subjects, closure of the hernia, assessments ofscarring and restoration of abdominal muscle function as assessed by aqualified clinician would be. Small biopsies of the repair can also betaken for direct assessment of muscle regeneration by histology by aqualified histotechnologist under the supervision of a clinician.However, it would be advisable to keep such invasive diagnoses to aminimum.

Methods

Provided herein are methods of promoting wound healing, organ or tissuereplacement and tissue regeneration following injury, disease, surgeryor congenital malformation in a subject, comprising administering to thesubject one or more of the herein provided compositions (e.g., tissueengineered units with polypeptides, nucleic acids, or vectors orcombinations of said units in three and two dimensional arrays).

The provided method can reduce scar tissue formation in a subjectfollowing tissue injury. By “scar tissue” is meant the fibrous(fibrotic) connective tissue that forms at the site of injury or diseasein any tissue of the body, caused by the overproduction of disorganizedcollagen and other connective tissue proteins, which acts to patch thebreak in the tissue. Scar tissue may replace injured skin and underlyingmuscle, damaged heart muscle, or diseased areas of internal organs suchas the liver. Dense and thick, it is usually paler than the surroundingtissue because it is poorly supplied with blood, and although itstructurally replaces destroyed tissue, it cannot perform the functionsof the missing tissue. It is composed of collagenous fibers, which willoften restrict normal elasticity in the tissue involved. Scar tissue maytherefore limit the range of muscle movement or prevent propercirculation of fluids when affecting the lymphatic or circulatorysystem. Glial scar tissue following injury to the brain or spinal chordis one of the main obstacles to restoration of neural function followingdamage to the central nervous system. A reduction in scar tissue can beassessed by the population of cell types within the injured site. Forexample, a reduction in glial scar tissue can be estimated by anincreased ratio of neuronal to astrocytic cells. A reduction in scartissue formation can be measured by a simple measurement of scar widthor area of scar tissue (Wilgus et al., 2003). In addition histologicalassessments can be made about the restoration of structural complexitywithin healed tissue in comparison to normal tissue. The reduction inscar tissue can be partial or complete, meaning 10, 20, 30, 40, 50, 60,70, 80, 90, 100% reduction, or any amount of reduction in between ascompared to native or control levels.

In addition to reducing fibrotic tissue formation in a subject infollowing tissue injury, the provided compositions and methods can alsobe used to treat disorders associated with pathological increases infibrotic tissue formation in a subject, such as for example, psoriasis,cutaneous and systemic mastocytosis, asthma, eczema, sinusitis,atherosclerosis, rheumatoid arthritis, inflammatory bowel disease,multiple sclerosis, pulmonary fibrosis and cystic fibrosis. A reductionin fibrotic tissue formation in a subject can be measured by clinicaljudgment of a doctor assessing whether a regain in normal structure andfunction of a given tissue and/or organ in a subject has resultedfollowing a treatment. As an example, for psoriasis a doctor wouldassess the subject's skin to determine whether there has been areduction in patches of raised red skin covered by flaky white buildup.Certain kinds of psoriasis, are characterized by a pimple-ish (pustularpsoriasis) or burned (erythrodermic) appearance. In such cases, thedoctor would determine whether treatment has resulted in the reductionof these symptoms. The reduction in fibrotic tissue can be partial orcomplete, meaning 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, orany amount of reduction in between as compared to native or controllevels. In the case of an tissue or organ in which a subject where adoctor judges that a biopsy is clinically available and/or necessary orin an animal model of the human disease, tissue fragments of biopsieswould be prepared and tissue histological structure would be assessed bya clinical pathologist and/or trained histopathologist to determine ifreduction in fibrosis and restoration of normal tissue structure andfunction has occurred. The area of fibrosis to normal tissue can also bequantitatively assessed on such histological preparations.

The provided method can improve tissue regeneration following tissueinjury in a subject. By “regeneration” is meant the renewal, re-growth,or restoration of a body or a bodily part, tissue, or substance afterinjury or as a normal bodily process. In contrast to scarring, tissueregeneration involves the restoration of the tissue to its originalstructural, functional, and physiological condition. This is alsoreferred to herein as tissue “complexity”. The restoration can bepartial or complete, meaning 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%restoration, or any amount of restoration in between as compared tonative or control levels. As an example, in the case of a skin injury,tissue regeneration can involve the restoration of hair follicles,glandular structures, blood vessels, muscle, or fat. In the case of abrain injury, tissue regeneration can involve maintenance or restorationof neurons. As an example in the case of skin an improvement in tissueregeneration can be assessed by measurements of the volume of fibrousscar tissue to normal regenerated skin as a ratio. As another example,counts can be made of discrete regenerating structures such asregenerating skin glands normalized to the volume of the wound area. Asanother example, counts of the density of cardiomyocytes can be made inthe area of heart normally comprised of scar tissue following thehealing of a myocardial infarction. Echocardiography can be used tomeasure the amount of recovery of cardiac function resulting from theregeneration of muscle cell in this scar tissue.

In one aspect, tissue regeneration involves the recruitment anddifferentiation of stem cells and/or progenitors cells to replace thedamaged cells. These stem cells can be generated from the exogenous stemcells comprising the tissue engineered composition or be endogenousprompted by the composition to join, fuse or otherwise combine in theregenerative repair process. As used herein, a “stem cell” is anundifferentiated cell found among differentiated cells in a tissue ororgan, or introduced as part of the tissue engineered composition asdescribed elsewhere herein. The primary roles of stem cells in a livingorganism are to maintain and repair the tissue in which they are found.By stem cell differentiation is meant the process whereby anunspecialized cell (e.g., stem cell) acquires the features of aspecialized cell such as a skin, neural, heart, liver, or muscle cell.As an example, in the case of a skin injury, tissue regeneration caninvolve the differentiation of stem cells present in the epithelium intohair follicles (Alonso and Fuchs, 2003). In the case of a brain injury,tissue regeneration can involve the differentiation of stem cells intoneurons. In the case of a cardiac injury, tissue regeneration caninvolve the differentiation of stem cells into cardiomyocytes of varioustypes (e.g., myocytes, conduction cells and nodal cells). The providedmethod can enhance stem cell differentiation following tissue injury ina subject. Enhanced stem cell differentiation can be measured byproviding a clinically acceptable genetic or other means of markingendogenous or engrafted stem cells and determining the frequency ofdifferentiation and incorporation of marked stem cells into normaltissue structures. The frequency of stem cell contribution to the repaircan be partial or complete, meaning 1, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, 100% contribution, or any amount of contribution in between ascompared to native or control levels. As another example, certainstructures such as hair follicles are known to be regenerated fromendogenous stem cells following tissue injury. As such, counts of markedstem cell derived hair follicles normalized to tissue injury area wouldserve as a quantitative assessment of enhanced stem celldifferentiation. In a further example, marked resident stem cells inskeletal or cardiac muscle will be prompted to the contribute to therepair process. In another example, counts of the density of stem cellderived cardiomyocytes can be made in the area of heart normallycomprised of scar tissue following the healing of a myocardialinfarction.

The provided composition can reduce inflammation in a subject. By“inflammation”, “inflammatory response” or “immune response” is meantthe reaction of living tissues to injury, infection or irritationcharacterized by redness, warmth, swelling, pain, and loss of function,produced as the result of increased blood flow and an influx of immunecells and secretions. Inflammation is the body's reaction to invadinginfectious microorganisms and results in an increase in blood flow tothe affected area, the release of chemicals that draw white blood cells,an increased flow of plasma, and the arrival of monocytes (or astrocytesin the case of the brain) to clean up the debris. Anything thatstimulates the inflammatory response is said to be inflammatory. Thus,in addition to reducing inflammation in a subject in response to tissueinjury, the provided compositions and methods can also be used to treatdisorders associated with pathological increases in levels ofinflammatory cells, including, for example, asthma, eczema, sinusitis,atherosclerosis, rheumatoid arthritis, inflammatory bowel disease,cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis.A reduction in inflammation can be measured by a reduction in thedensity of inflammatory cell types such as, for example, monocytes orastrocytes. A reduction in inflammation can be measured by a reductionin the density of inflammatory cell types such as, for example,neutrophils, macrophages, microglia, mast cells, basophils, andmonocytes. A measurement can be measured by reductions in allied cellssuch myofibroblasts and the like. A reduction in inflammation can becalculated by an in vivo measurement of neutrophil activity (Jones etal., 1994). In addition factors like frequency of mast celldegranulation or measurement of histamine levels or levels of reactiveoxygen species can be used as measurements of reduction in inflammation.The level of inflammation can also be indirectly measured by checkingfor transcription levels of certain genes by qRT-PCR for e.g. geneslike, Interferon-alpha, -beta and -gamma, Tumor Necrosis Factor-alpha,Interleukine 1beta, -2, -4, -5, -6, -8, -12, -18, -23, -27, CD4, CD28,CD80, CD86, MHCII, and iNOS. Measurement of pro-inflammatory cytokinelevels in the tissues and or bodily fluids of the subject includingplasma can measure a reduction in inflammation. It is noteworthy that amechanism of action may be by inhibition of inflammatory cell migrationand/or inhibition of pro-inflammatory chemicals (histamine, reactiveoxygen species) and pro-inflammatory cytokines such as Interleukin(IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF). The reduction ininflammation can be partial or complete, meaning 10, 20, 30, 40, 50, 60,70, 80, 90, 100% reduction, or any amount of reduction in between ascompared to native or control levels.

As used herein, tissue injury can result from, for example, a cut,scrape, compression wound, stretch injury, laceration wound, crushwound, bite wound, graze, bullet wound, explosion injury, body piercing,stab wound, surgical wound, surgical intervention, medical intervention,host rejection following cell, tissue or organ grafting, pharmaceuticaleffect, pharmaceutical side-effect, bed sore, radiation injury, cosmeticskin wound, internal organ injury, disease process (e.g., asthma,cancer), infection, infectious agent, developmental process,maturational process (e.g., acne), genetic abnormality, developmentalabnormality, environmental toxin, allergen, scalp injury, facial injury,jaw injury, sex organ injury, joint injury, excretory organ injury, footinjury, finger injury, toe injury, bone injury, eye injury, cornealinjury, muscle injury, adipose tissue injury, lung injury, airwayinjury, hernia, anus injury, piles, ear injury, skin injury, abdominalinjury, retinal injury, eye injury, corneal injury, arm injury, leginjury, athletic injury, back injury, birth injury, premature birthinjury, toxic bite, sting, tendon injury, ligament injury, heart injury,heart valve injury, vascular system injury, cartilage injury, lymphaticsystem injury, craniocerebral trauma, dislocation, esophagealperforation, fistula, nail injury, foreign body, fracture, frostbite,hand injury, heat stress disorder, laceration, neck injury, selfmutilation, shock, traumatic soft tissue injury, spinal cord injury,spinal injury, sprain, strain, tendon injury, ligament injury, cartilageinjury, thoracic injury, tooth injury, trauma, nervous system injury,burn wound, wind burn, sun burn, chemical burn, aging, aneurism, stroke,digestive tract injury, infarct, or ischemic injury.

It is understood that the disclosed compositions, devices and methodsare not limited to the particular methodology, protocols, and reagentsdescribed as these can vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of such polypeptides, reference to “thepeptide” is a reference to one or more peptides and equivalents thereofknown to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges can be expressed herein as from “˜” and “about” one particularvalue, and/or to “˜” and “about” another particular value. When such arange is expressed, also specifically contemplated and considereddisclosed is the range from the one particular value and/or to the otherparticular value unless the context specifically indicates otherwise.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another, specifically contemplated embodiment that should beconsidered disclosed unless the context specifically indicatesotherwise. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, andindependently of the other endpoint unless the context specificallyindicates otherwise. Finally, it should be understood that all of theindividual values and sub-ranges of values contained within anexplicitly disclosed range are also specifically contemplated and shouldbe considered disclosed unless the context specifically indicatesotherwise. The foregoing applies regardless of whether in particularcases some or all of these embodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

“Pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

“Promote,” “promotion,” and “promoting” refer to an increase in anactivity, response, condition, disease, or other biological parameter.This can include but is not limited to the initiation of the activity,response, condition, or disease. This can also include, for example, a10% increase in the activity, response, condition, or disease ascompared to the native or control level. Thus, the increase can be a 10,20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of increase inbetween as compared to native or control levels.

As used herein, “inhibit,” “inhibiting,” “inhibition” and “loss offunction” mean to decrease an activity, response, condition, disease, orother biological parameter. This can include, but is not limited to, thecomplete loss of activity, response, condition, or disease. This canalso include, for example, a 10% reduction in the activity, response,condition, or disease as compared to the native or control level. Thus,the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or anyamount of reduction in between as compared to native or control levels.

As used herein, agonizing, activating or gain of function mean toincrease an activity, response, condition, disease, or other biologicalparameter. This can also include, for example, a 10% increase in theactivity, response, condition, or disease as compared to the native orcontrol level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70,80, 90, 100%, 200%, 400% or any amount of increase in between ascompared to native or control levels.

By “treat” or “treatment” is meant a method of reducing the effects of adisease or condition. Treatment can also refer to a method of reducingthe underlying cause of the disease or condition itself rather than justthe symptoms. The treatment can be any reduction from native levels andcan be but is not limited to the complete ablation of the disease,condition, or the symptoms of the disease or condition. For example, adisclosed method for promoting wound healing is considered to be atreatment if there is a 10% reduction in one or more symptoms of thedisease in a subject with the disease when compared to native levels inthe same subject or control subjects. Thus, the reduction can be a 10,20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

As used herein, “subject” includes, but is not limited to, animals,plants, bacteria, viruses, parasites and any other organism or entitythat has nucleic acid. The subject can be a vertebrate, morespecifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a birdor a reptile or an amphibian. The subject can be an invertebrate, morespecifically an arthropod (e.g., insects and crustaceans). The term doesnot denote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Apatient refers to a subject afflicted with a disease or disorder. Theterm “patient” includes human and veterinary subjects.

What is claimed:
 1. A kit comprising: primed living bone-marrow stromalcells joined to and at least partially within a three-dimensionalflexible hydrogel structure, the hydrogel structure comprising collagenand having a toroid shape; and an isolated polypeptide comprising thecarboxy-terminal amino acid sequence of an alpha Connexin protein, or aconservative variant thereof, wherein the polypeptide does not comprisethe full length alpha Connexin protein and the carboxy-terminal aminoacid sequence is capable of inhibiting gap junction formation.
 2. Amethod of forming a toroid, hydrogel structure comprising placing primedliving bone marrow stromal cells on a three-dimensional, flexiblehydrogel structure, the hydrogel structure comprising collagen, andpermitting epithelialization of the cells in which the cells attach tothe surface of the hydrogel structure, undergo epithelial mesenchymaltransition and penetrate the surface of the hydrogel structure to form atoroid shape.